IL2 agonists and methods of use thereof

ABSTRACT

The present disclosure relates to IL2 agonists with improved therapeutic profiles.

1. CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/127,359, which claims the priority benefit of U.S. provisionalapplication No. 62/951,831, filed Dec. 20, 2019, the contents of each ofwhich are incorporated herein in their entireties by reference thereto.

2. SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 18, 2020, isnamed RGN-006C-US_SL and is 159,460 bytes in size.

3. BACKGROUND

Interleukin 2 (IL-2 or IL2) is a pluripotent cytokine produced primarilyby CD4⁺ helper T cells. It stimulates the proliferation anddifferentiation of T cells, induces the generation of cytotoxic Tlymphocytes (CTLs) and the differentiation of peripheral bloodlymphocytes to cytotoxic cells and lymphokine-activated killer (LAK)cells, promotes cytokine and cytolytic molecule expression by T cells,facilitates the proliferation and differentiation of B-cells and thesynthesis of immunoglobulin by B-cells, and stimulates the generation,proliferation and activation of natural killer (NK) cells (see Waldmann,2009, Nat Rev Immunol 6:595-601 and Malek, 2008, Annu Rev Immunol26:453-79).

IL2 has three different receptors: the high affinity receptor, theintermediate affinity receptor, and the low affinity receptor. The highaffinity receptor has three subunits: the interleukin 2 receptor alpha(IL-2Rα; CD25), the interleukin 2 receptor beta (IL-2Rβ; CD122), and theinterleukin 2 receptor gamma (IL-2Rγ; CD132; common gamma chain). Thelow affinity receptor is IL-2Rα, which is a 55 kD polypeptide (p55) thatappears upon T cell activation and was originally called Tac (for Tactivation) antigen. IL-2Rα binds IL2 with a K_(D) of approximately 10⁻⁸M. Binding of IL2 to cells expressing only IL-2Rα does not lead to anydetectable biologic response.

The intermediate affinity receptor is composed of IL-2Rβ and IL-2Rγ.IL-2Rβ is a member of the type I cytokine receptor family characterizedby the two cysteine/WSXWS motif (SEQ ID NO:1). IL-2Rγ, a 64 kDpolypeptide, is also known as the common gamma chain because it isshared among a number of cytokine receptors, including the receptor forinterleukin-4 and interleukin-7. The intermediate affinity receptor alsomediates interleukin 15 (IL-15 or IL15) signaling.

Resting immune cells are thought to express only the intermediateaffinity receptor. Upon antigen receptor-mediated immune cellactivation, e.g., of resting T cells, IL-2Rα is rapidly expressed. OnceIL-2Rα binds IL2, it then sequentially engages IL-2Rβ and IL-2Rγ. IL2binding by the IL-2Rαβγ complex results in signal transduction through aSTAT5 and IL2 mediated growth stimulation of, inter alia, effector Tcells that destroy virus-infected cells and tumor cells.

In addition to its stimulatory effects on effector T cells, IL2 mediatesactivation-induced cell death (AICD) in T cells (Lenardo et al., 1991,Nature 353:858-61). AICD is a process by which fully activated T cellsundergo programmed cell death, resulting in tolerance not only toestablished normal self-antigens but also to persistent antigens such astumor antigens.

IL2 is also involved in the maintenance of peripheral CD4⁺ CD25⁺regulatory T (T_(reg)) cells (see, e.g., Fontenot et al., 2005, NatureImmunol 6:1142-51), which are also known as suppressor T cells. Theysuppress effector T cells from destroying their (self-)target, eitherthrough cell-cell contact by inhibiting T cell help and activation, orthrough release of immunosuppressive cytokines such as IL-10 or TGFβ.Depletion of T_(reg) cells was shown to enhance IL2-induced anti-tumorimmunity (Imai et al., 2007, Cancer Sci 98:416-23).

Due to its pleotropic effects, IL2 is not optimal for inhibiting tumorgrowth. The use of IL2 as an antineoplastic agent has been limited bythe serious toxicities that accompany the doses necessary for a tumorresponse. Proleukin® (marketed by Prometheus Laboratories, San Diego,Calif.), is a recombinant form of IL2 that is approved for the treatmentof metastatic melanoma and metastatic renal cancer, but its side effectsare so severe that its use is only recommended in a hospital settingwith access to intensive care. Patients receiving high-dose IL2treatment frequently experience severe cardiovascular, pulmonary, renal,hepatic, gastrointestinal, neurological, cutaneous, haematological andsystemic adverse events, which require intensive monitoring andin-patient management. The major side effect of IL2 therapy is vascularleak syndrome (VLS), which leads to the accumulation of interstitialfluid in the lungs and liver resulting in pulmonary edema and liverdamage. There is no treatment for VLS other than withdrawal of IL2.Low-dose IL2 regimens have been tested in patients to avoid VLS,however, at the expense of suboptimal therapeutic results. It has beenshown that IL2-induced pulmonary edema resulted from direct binding ofIL2 to lung endothelial cells, which express low to intermediate levelsof functional high affinity IL2 receptors (Krieg et al., 2010, Proc NatAcad Sci USA 107:11906-11).

A variety of IL2 variants have been generated with the aim of reducingthe toxicity of IL2 cancer therapy. The prevailing approach is thegeneration of IL2 molecules that allow IL2 binding to the intermediateaffinity receptor but disfavor the association of IL2 with CD25 (thustermed CD122-biased). The rationale for this approach is two-fold.First, CD25 is prominently expressed on T_(reg) cells, where the excessof CD25 could serve as a sink, thereby taking IL2 away from effector Tcells. Second, endothelial cells, which mediate VLS, express CD25. Intheory, reducing binding to CD25 (IL-2Rα) could redirect the IL2 signalto effector T cells, improving anti-tumor efficacy, and reduce VLS,thereby reducing toxicity. Strategies to obtain CD122-biased IL2formulations include, for example, CD122-directed IL2 complexes, inwhich an anti-IL2 monoclonal antibody covers the CD25-binding site, IL2muteins with a mutation at the CD25-binding site, and IL2 carryingpolyethylene glycol (PEG) groups at the CD25-binding site. Onur andArenas-Ramirez, 2019, Swiss Med Wkly 149: w14697.

Despite the general acceptance in the field of CD122 directed IL2therapies being developed for cancer therapy, it has been surprisinglydiscovered that such molecules have poor therapeutic indices for cancertherapy, with high, toxic doses required to confer modest anti-cancereffects.

Thus, there is a need in the art for novel IL2 therapies with improvedtherapeutic efficacy and safety profiles.

4. SUMMARY

The present disclosure stems from a number of discoveries regarding theactivities of IL2 molecules that impact anti-tumor efficacy. As shownherein, CD122 directed IL2 molecules have surprisingly only modestanti-tumor efficacy, even at high doses that result in apparenttoxicity. This can be explained by a number of other discoveries. First,the inventors have discovered that while CD122 directed IL2 moleculeswere designed to preferentially expand NK and CD8⁺ T cells relative toTreg cells, they primarily do so in the blood and spleen but to a muchlesser extent in tumors, in contrast to wild IL2 preferentially expandsCD8⁺ T cells in the tumor but not in the periphery. In addition, CD8⁺ Tcells expanded by CD122-biased IL2 molecules are mostly CD44⁺CD62L⁺central memory-like T cells and lack specificity against tumor cells,whereas wild type IL2 can expand CD44⁺CD62L⁻ effector CD8⁺ T cells.Thus, the anti-tumor efficacy of these molecules is reduced relative towild type IL2. Further, the inventors have discovered that tumor Tregcells are less responsive to IL2 than blood and spleen Treg cells. Thus,the adverse effects of non-CD122 directed forms of IL2 cancer therapycan be mitigated by directing such non-CD122 directed forms of IL2 totumors, where Tregs are less responsive to IL2 and IL2 preferentiallyexpands CD8+ T cells.

Further, the inventors have discovered that the therapeutic indices of avariety of IL2 molecules with various degrees of receptor attenuationcan be improved by appropriately balancing the ability of an IL2 agonistto be localizated to a site or cell type of interest (e.g., the tumorenvironment generally or tumor reactive T cells specifically) andmodulating the level activity of the IL2 component at the site ofinterest and/or until it reaches the site or cell type of interest.

Based on the foregoing discoveries, the inventors have developed IL2molecules (referred to herein as IL2 agonists) that are believed to bemore effective than the CD122 directed IL2 molecules currently underdevelopment for the treatment of cancer. The IL2 agonists of thedisclosure are not directed to CD122 and, in some instances, havereduced or minimized CD122 binding. The IL2 agonists of the disclosurehave an IL2 moiety and (1) an optional tumor targeting moiety and/or (2)an optional multimerization, e.g., dimerization, moiety and/or (3) anoptional stabilization moiety. The tumor targeting moiety, e.g., anantigen binding domain (“ABD”) of an antibody, can, for example, bind toa target molecule present on the tumor surface (e.g., a tumor associatedantigen), the tumor microenvironment, and/or tumor reactive lymphocytes.

IL2 moieties that can be used in the IL2 agonists of the disclosure aredescribed in Section 6.3.

Targeting moieties that can be used in the IL2 agonists of thedisclosure are described in Section 6.4.

Multimerization moieties that can be used in the IL2 agonists of thedisclosure are described in Section 6.5.

Stabilization moieties that can be used in the IL2 agonists of thedisclosure are described in Section 6.6.

Various exemplary configurations of the IL2 agonists of the disclosureare described in specific embodiments 1 to 166, 199 to 258 and 267 to322, infra.

Linkers that can be used to connect different components of the IL2agonists of the disclosures are described in Section 6.7.

The disclosure further provides nucleic acids encoding the IL2 agonistsof the disclosure. The nucleic acids encoding the IL2 agonists can be asingle nucleic acid (e.g., a vector encoding all polypeptide chains ofan IL2 agonist) or a plurality of nucleic acids (e.g., two or morevectors encoding the different polypeptide chains of an IL2 agonist).The disclosure further provides host cells and cell lines engineered toexpress the nucleic acids and IL2 agonists of the disclosure. Thedisclosure further provides methods of producing an IL2 agonist of thedisclosure. Exemplary nucleic acids, host cells, and cell lines, andmethods of producing an IL2 agonist are described in Section 6.8 andspecific embodiments 167 to 169, 259 to 261 and 323 to 325 infra.

The disclosure further provides pharmaceutical compositions comprisingthe IL2 agonists of the disclosure. Exemplary pharmaceuticalcompositions are described in Section 6.9 and specific embodiments 170,262 and 326 infra.

Further provided herein are methods of using the IL2 agonists and thepharmaceutical compositions of the disclosure, e.g., for treating cancerand immune disorders. Exemplary methods are described in Section 6.10.The IL2 agonists of the disclosure are useful in combination therapy,for example as an adjunct to CART therapy. Exemplary combination therapymethods are disclosed in 6.11. Specific embodiments of the methods oftreatment of the disclosure are described in specific embodiments 171 to198, 263 to 266 and 327 to 346 infra.

5. BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 illustrates the IL2 and IL15 signaling pathways, which haveunique receptor subunits but share common β/γ receptor subunits.

FIGS. 2A-2D illustrate presentation of antigens by antigen-presentingcells to T cells via the class I (FIG. 2A) and class II (FIG. 2B) MHCcomplexes, and activation of the T cell receptor complexes byMHC-peptide complexes (FIGS. 2C-2D).

FIGS. 3A-3B show the equilibrium between different structures formedfrom two different species of IL-2Rα-containing IL2 muteins.

FIGS. 4A-4C show activity of IL2M1 on human PBMCs. IL2M1 has reducedactivity on IL-2Rα+ Tregs (FIG. 4A), whereas it maintains the fullactivity on IL-2Rα− CD8+ T cells (FIG. 4B) and NK cells (FIG. 4C).

FIGS. 5A-5D show selective expansion of NK (FIG. 5B) and CD8+ T cells(FIG. 5C-5D) relative to Tregs (FIG. 5A) by IL2M1 in vivo, whereas IL2M0(referred to as IL2-Fc in figure) preferentially expands Tregs (FIGS.5A-5D).

FIGS. 6A-6B show average tumor volumes (mm³+SD) (FIG. 6A) andKaplan-Meier survival curves (FIG. 6B) of mice treated with IL2 muteinsand/or anti-PD1 antibodies.

FIGS. 7A-7C.5 show that IL2M0 (referred to as IL2-Fc in figure) inducesantitumor response in a dose dependent manner.

FIGS. 8A-8C show modest antitumor efficacy (FIG. 8A, 8B) and concomitantapparent toxicity (FIG. 8C) for IL2M1.

FIGS. 9A-9C.4 show modest antitumor efficacy (FIG. 9A) and concomitantapparent toxicity (FIG. 9B) for IL15M1, as well as the peripheral immunecell profile induced by IL15M1 (FIGS. 9C.1-C.4).

FIGS. 10A.1-10R show activity of IL2M0 (referred to as IL2-Fc in figure)and IL2M1 on proliferation of intratumoral and peripheral lymphocytes.

FIGS. 11A-11B show differential IL2 responsiveness of splenic andtumor-infiltrating Tregs and CD8+ T cells.

FIGS. 12A-12D show that IL2M2 and IL2M3 exhibit attenuated activityacross multiple lymphocyte populations in human PBMCs (FIG. 12A-FIG.12C) and reduced in vivo toxicity (FIG. 12D). The results in FIG. 12Athrough FIG. 12C are obtained with IL2M2 and IL2M3 protein and in FIG.12D are based on in vivo administration of IL2M2 and IL2M3 encodingnucleic acids by hydrodynamic DNA delivery (“HDD”).

FIGS. 13A-13C show that IL2M2 displays anti-tumor efficacy as a singleagent in multiple syngeneic mouse tumor models. IL2M2 was delivered viahydrodynamic DNA delivery (HDD) (FIGS. 13A and 13B) or as purifiedprotein (FIG. 13C).

FIGS. 14A-14C show activity of IL2M4 and IL2M5 on different lymphocytepopulations of human PBMC.

FIGS. 15A-15D show antitumor efficacy of IL2M5.

FIGS. 16A-16B show the results of SEC-MALS studies of IL2M2 (FIG. 16A),which primarily consists of higher order oligomers, vs. IL2M3 (FIG.16B), which predominantly exists as a Fc homodimer, believed to be aresult of the orientation of the IL2 domain relative to the Fc domainand/or length of the linker connecting the domains.

FIG. 17 shows results of FACS binding of anti-PD1 and T1-IL2M3 toHEK293-mPD1 cells.

FIGS. 18A-18E.2 show superior anti-tumor efficacy of T1-IL2M3 to thecombination of anti-PD1 and non-targeted IL2M3 (FIGS. 18A-18B.4), andthat T1-IL2M3 expands effector CD8+ T cells that are PD1+ while causesless expansion of Tregs than non-targeted IL2M3 (FIGS. 18C.1-18E.2).

FIG. 19 shows a schematic of (a) IL2 binding by the IL-2Rαβγ complex,resulting in signal transduction through STAT5 and strong cellactivation (left portion of figure) (b) low level binding of ananti-PD1-IL2 Mutein 3 fusion to the IL-2Rαβγ complex on lymphocytes andendothelial cells in the absence of cell surface expressed PD-1,resulting in low or no activation of those cells (middle portion of thefigure between the two dotted lines), and (c) binding of an anti-PD1-IL2Mutein 3 fusion to the IL-2Rαβγ complex on tumor specific T-cells withcell surface expressed PD-1, resulting in sustained signaling and T-cellactivation.

FIGS. 20A-20D show anti-tumor efficacy of an anti-mPD1-IL2 mutein 3fusion in murine syngeneic tumor models: lung (FIG. 20A); skin (FIG.20B); breast (FIG. 20C); and colon (FIG. 20D).

FIGS. 21A-21B show anti-tumor efficacy of an anti-LAG3-IL2 Mutein 3fusion (FIG. 21A) and anti-tumor efficacy of an anti-LAG3-IL2 Mutein 3fusion in combination with anti-mPD1 antibody (FIG. 21B).

FIGS. 22A.1-22B show homodimeric and heterodimeric embodiments of singlechain peptide-MHC-targeted IL2 muteins (FIGS. 22A.1-22A.2) and resultsfrom a study demonstrating that a single chain peptide-MHC-targeted IL2mutein fusion allows selective stimulation of antigen-specific mouseCD8+ T cells (FIG. 22B).

FIGS. 23A-23B show the activity of single chain peptide-MHC targeted IL2muteins on non-CD8+ T cells that do not express antigen-specific TCR.The different muteins show indistinguishable activities.

FIG. 24 shows selective stimulation of CMV antigen-specific human CD8+ Tcells by a single chain IL2 mutein with a peptide-MHC targeting moiety.

FIGS. 25A-25C show a schematic of a chimeric antigen receptor (CAR)comprising a VL-VH scFv recognizing the peptide-MHC targeting moietyreferred to herein as T3, a human CD8a hinge and transmembrane (TM)domain, a 4-1BB costimulatory domain, a CD3z signaling domain, and aP2A:eGFP sequence for tracking CAR-transduced T cells (FIG. 25A) and thefrequency and composition of viable CAR-T cells post-expansion (FIGS.25B-25C).

FIGS. 26A-26B illustrate the structures of the bivalent targeted IL2muteins T2-IL2M6 and T3-IL2M6 (FIG. 26A) and the monovalent targeted IL2muteins T7-IL2M7 and T8-IL2M7 (FIG. 26B), both with peptide-MHCtargeting moieties.

FIGS. 27A-27B show STAT5 stimulation of CAR-expressing CD4+(FIG. 27A)and CD8+(FIG. 27B) T cells by the peptide-MHC targeted IL2 muteinsillustrated in FIGS. 26A and 26B.

FIGS. 28A.1-28C show selective enrichment of CAR-T by a monovalent IL2mutein with a targeting moiety recognized by the scFV of the CAR. FIGS.28A.1-28A.16 shows the frequency of CAR-T cells is enriched duringexpansion in response to monovalent single chain peptide loaded HLA-A2as determined by flow cytometry. All biologics were maintained at aconcentration of 3.3×10⁻¹⁰M. FIGS. 28B.1-28B.4 shows that theCAR^(Pos)/CAR^(Neg) ratio selectively increases during expansion inresponse to monovalent single chain peptide loaded HLA-A2 fused toattenuated IL(2m) (open circle). Recombinant IL2 (Aldesleukin) yieldsthe largest number of total T cells yet the degree of CAR-T enrichmentis lesser relative to single chain peptide loaded HLA-A2 fused toattenuated IL2(2m). FIG. 28C shows the expansion of CAR-T using singlechain peptide loaded HLA-A2 fused to attenuated IL2(2m) (open circle)yields the greatest number of viable CAR-T.

FIG. 29 shows the results of an in vivo PK assessment of monovalent andbivalent targeted attenuated IL2 muteins in immunocompetent (C57BL/6J)and immunodeficient (NOD.Scid.IL2Rgnull) mice. Both bivalent andmonovalent muteins show delayed clearance relative to hIL2 fused to Fc(IL2-Fc). Mice were injected with each biologic and blood samples werecollected at 2, 24, 48, and 72 hr after dosing. Pharmacokinetics of Fcfusion proteins in plasma was investigated by Western blot. The proteinsof interest are denoted by the black boxes.

6. DETAILED DESCRIPTION 6.1. Definitions

About, Approximately: The terms “about”, “approximately” and the likeare used throughout the specification in front of a number to show thatthe number is not necessarily exact (e.g., to account for fractions,variations in measurement accuracy and/or precision, timing, etc.). Itshould be understood that a disclosure of “about X” or “approximately X”where X is a number is also a disclosure of “X.” Thus, for example, adisclosure of an embodiment in which one sequence has “about X %sequence identity” to another sequence is also a disclosure of anembodiment in which the sequence has “X % sequence identity” to theother sequence.

And, or: Unless indicated otherwise, an “or” conjunction is intended tobe used in its correct sense as a Boolean logical operator, encompassingboth the selection of features in the alternative (A or B, where theselection of A is mutually exclusive from B) and the selection offeatures in conjunction (A or B, where both A and B are selected). Insome places in the text, the term “and/or” is used for the same purpose,which shall not be construed to imply that “or” is used with referenceto mutually exclusive alternatives.

Antigen Binding Domain or ABD: The term “antigen binding domain” or“ABD” as used herein refers to the portion of a targeting moiety that iscapable of specific, non-covalent, and reversible binding to a targetmolecule.

Associated: The term “associated” in the context of an IL2 agonist or acomponent thereof (e.g., a targeting moiety such as an antibody) refersto a functional relationship between two or more polypeptide chains. Inparticular, the term “associated” means that two or more polypeptidesare associated with one another, e.g., non-covalently through molecularinteractions or covalently through one or more disulfide bridges orchemical cross-linkages, so as to produce a functional IL2 agonist.Examples of associations that might be present in an IL2 agonist of thedisclosure include (but are not limited to) associations betweenhomodimeric or heterodimeric Fc domains in an Fc region, associationsbetween VH and VL regions in a Fab or scFv, associations between CH1 andCL in a Fab, and associations between CH3 and CH3 in a domainsubstituted Fab.

Bivalent: The term “bivalent” as used herein in reference to an IL2moiety and/or a targeting moiety in an IL2 agonist means an IL2 agonistthat has two IL2 moieties and/or targeting moieties, respectively.Typically, IL2 agonists that are bivalent for an IL2 moiety and/or atargeting moiety are dimeric (either homodimeric or heterodimeric).

Cancer: The term “cancer” refers to a disease characterized by theuncontrolled (and often rapid) growth of aberrant cells. Cancer cellscan spread locally or through the bloodstream and lymphatic system toother parts of the body. Examples of various cancers are describedherein and include but are not limited to, breast cancer, prostatecancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer,colorectal cancer, renal cancer, liver cancer, brain cancer, adrenalgland cancer, autonomic ganglial cancer, biliary tract cancer, bonecancer, endometrial cancer, eye cancer, fallopian tube cancer, genitaltract cancers, large intestinal cancer, cancer of the meninges,oesophageal cancer, peritoneal cancer, pituitary cancer, penile cancer,placental cancer, pleura cancer, salivary gland cancer, small intestinalcancer, stomach cancer, testicular cancer, thymus cancer, thyroidcancer, upper aerodigestive cancers, urinary tract cancer, vaginalcancer, vulva cancer, lymphoma, leukemia, lung cancer and the like.

Complementarity Determining Region or CDR: The terms “complementaritydetermining region” or “CDR,” as used herein, refer to the sequences ofamino acids within antibody variable regions which confer antigenspecificity and binding affinity. In general, there are three CDRs ineach heavy chain variable region (CDR-H1, CDR-H2, HCDR-H3) and threeCDRs in each light chain variable region (CDR1-L1, CDR-L2, CDR-L3).Exemplary conventions that can be used to identify the boundaries ofCDRs include, e.g., the Kabat definition, the Chothia definition, theABM definition and the IMGT definition. See, e.g., Kabat, 1991,“Sequences of Proteins of Immunological Interest,” National Institutesof Health, Bethesda, Md. (Kabat numbering scheme); Al-Lazikani et al.,1997, J. Mol. Biol. 273:927-948 (Chothia numbering scheme); Martin etal., 1989, Proc. Natl. Acad. Sci. USA 86:9268-9272 (ABM numberingscheme); and Lefranc et al., 2003, Dev. Comp. Immunol. 27:55-77 (IMGTnumbering scheme). Public databases are also available for identifyingCDR sequences within an antibody.

EC50: The term “EC50” refers to the half maximal effective concentrationof a molecule (such as an IL2 agonist) which induces a response halfwaybetween the baseline and maximum after a specified exposure time. TheEC50 essentially represents the concentration of an antibody or IL2agonist where 50% of its maximal effect is observed. In certainembodiments, the EC50 value equals the concentration of an IL2 agonistthat gives half-maximal STAT5 activation in an assay as described inSection 7.1.2.

Epitope: An epitope, or antigenic determinant, is a portion of anantigen (e.g., target molecule) recognized by an antibody or otherantigen-binding moiety as described herein. An epitope can be linear orconformational.

Fab: The term “Fab” in the context of a targeting moiety of thedisclosure refers to a pair of polypeptide chains, the first comprisinga variable heavy (VH) domain of an antibody N-terminal to a firstconstant domain (referred to herein as C1), and the second comprisingvariable light (VL) domain of an antibody N-terminal to a secondconstant domain (referred to herein as C2) capable of pairing with thefirst constant domain. In a native antibody, the VH is N-terminal to thefirst constant domain (CH1) of the heavy chain and the VL is N-terminalto the constant domain of the light chain (CL). The Fabs of thedisclosure can be arranged according to the native orientation orinclude domain substitutions or swaps on that facilitate correct VH andVL pairings. For example, it is possible to replace the CH1 and CLdomain pair in a Fab with a CH3-domain pair to facilitate correctmodified Fab-chain pairing in heterodimeric molecules. It is alsopossible to reverse CH1 and CL, so that the CH1 is attached to VL and CLis attached to the VH, a configuration generally known as Crossmab.

Fc Domain and Fc Region: The term “Fc domain” refers to a portion of theheavy chain that pairs with the corresponding portion of another heavychain. The term “Fc region” refers to the region of antibody-basedbinding molecules formed by association of two heavy chain Fc domains.The two Fc domains within the Fc region may be the same or differentfrom one another. In a native antibody the Fc domains are typicallyidentical, but one or both Fc domains might advantageously be modifiedto allow for heterodimerization, e.g., via a knob-in-hole interaction.

Host cell: The term “host cell” as used herein refers to cells intowhich a nucleic acid of the disclosure has been introduced. The terms“host cell” and “recombinant host cell” are used interchangeably herein.It is understood that such terms refer to the particular subject celland to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein. Typical host cells are eukaryotic host cells, such asmammalian host cells. Exemplary eukaryotic host cells include yeast andmammalian cells, for example vertebrate cells such as a mouse, rat,monkey or human cell line, for example HKB11 cells, PER.C6 cells, HEKcells or CHO cells.

IL2 Mutein: Is a variant IL2 molecule that has IL2 activity. The variantcan be an IL2 fusion protein (e.g., an IL2 fused to IL-2Rα) and/or amutant IL2, e.g., with one or more amino acid substitutions compared towild type IL2. An IL2 mutein can have altered function (e.g., receptorbinding, affinity, cytokine activity) and/or altered pharmacokinetics ascompared to wild type IL2. While in the context of the IL2 agonists ofthe disclosure, the term “IL2 mutein” sometimes refers to thenon-targeting components of the an IL2 molecule (and associated linkermoieties), and it is to be understood that the term “IL2 mutein”encompass IL2 molecules with or without a targeting moiety and with orwithout a multimerization moiety unless the context dictates otherwise.

Major histocompatibility complex and MHC: These terms refer to naturallyoccurring MHC molecules, individual chains of MHC molecules (e.g., MHCclass I α (heavy) chain, β2 microglobulin, MHC class II α chain, and MHCclass II β chain), individual subunits of such chains of MHC molecules(e.g., α1, α2, and/or α3 subunits of MHC class I α chain, α1-α2 subunitsof MHC class II α chain, β1-β2 subunits of MHC class II β chain) as wellas portions (e.g., the peptide-binding portions, e.g., thepeptide-binding grooves), mutants and various derivatives thereof(including fusions proteins), wherein such portion, mutants andderivatives retain the ability to display an antigenic peptide forrecognition by a T-cell receptor (TCR), e.g., an antigen-specific TCR.An MHC class I molecule comprises a peptide binding groove formed by theα1 and α2 domains of the heavy a chain that can stow a peptide of around8-10 amino acids. Despite the fact that both classes of MHC bind a coreof about 9 amino acids (e.g., 5 to 17 amino acids) within peptides, theopen-ended nature of MHC class II peptide binding groove (the α1 domainof a class II MHC a polypeptide in association with the β1 domain of aclass II MHC β polypeptide) allows for a wider range of peptide lengths.Peptides binding MHC class II usually vary between 13 and 17 amino acidsin length, though shorter or longer lengths are not uncommon. As aresult, peptides may shift within the MHC class II peptide bindinggroove, changing which 9-mer sits directly within the groove at anygiven time. Conventional identifications of particular MHC variants areused herein. The terms encompass “human leukocyte antigen” or “HLA”.

Monovalent: The term “monovalent” as used herein in reference to an IL2moiety and/or a targeting moiety in an IL2 agonist means an IL2 agonistthat has only a single IL2 moiety and/or targeting moiety, respectively.Typically, IL2 agonists that are monovalent for the IL2 moiety and/ortargeting moiety are monomeric or heterodimeric.

Operably linked: The term “operably linked” as used herein refers to afunctional relationship between two or more regions of a polypeptidechain in which the two or more regions are linked so as to produce afunctional polypeptide, or two or more nucleic acid sequences, e.g., toproduce an in-frame fusion of two polypeptide components or to link aregulatory sequence to a coding sequence.

Peptide-MHC complex, pMHC complex, peptide-in-groove: An (i) an MHCdomain (e.g., a human MHC molecule or portion thereof (e.g., thepeptide-binding groove thereof and e.g., the extracellular portionthereof), (ii) an antigenic peptide, and, optionally, (iii) a β2microglobulin domain (e.g., a human β2 microglobulin or portionthereof), where the MHC domain, the antigenic peptide and optional β2microglobulin domain are complexed in such a manner that permitsspecific binding to a T-cell receptor. In some embodiments, a pMHCcomplex comprises at least the extracellular domains of a human HLAclass I/human β2 microglobulin molecule and/or a human HLA class IImolecule.

Single Chain Fv or scFv: The term “single chain Fv” or “scFv” as usedherein refers to a polypeptide chain comprising the VH and VL domains ofantibody, where these domains are present in a single polypeptide chain.

Specifically (or selectively) binds: The term “specifically (orselectively) binds” as used herein means that a targeting moiety, e.g.,an antibody, or antigen binding domain (“ABD”) thereof, forms a complexwith a target molecule that is relatively stable under physiologicconditions. Specific binding can be characterized by a K_(D) of about5×10⁻²M or less (e.g., less than 5×10⁻²M, less than 10⁻²M, less than5×10⁻³M, less than 10⁻³M, less than 5×10⁻⁴M, less than 10⁻⁴M, less than5×10⁻⁵M, less than 10⁻⁵M, less than 5×10⁻⁶M, less than 10⁻⁶M, less than5×10⁻⁷M, less than 10⁻⁷M, less than 5×10⁻⁸M, less than 10⁻⁸M, less than5×10⁻⁹M, less than 10⁻⁹M, or less than 10⁻¹⁰M). Methods for determiningthe binding affinity of an antibody or an antibody fragment, e.g., anIL2 agonist or a component targeting moiety, to a target molecule arewell known in the art and include, for example, equilibrium dialysis,surface plasmon resonance (e.g., Biacore assays), fluorescent-activatedcell sorting (FACS) binding assays and the like. An IL2 agonist of thedisclosure comprising a targeting moiety or an ABD thereof thatspecifically binds a target molecule from one species can, however, havecross-reactivity to the target molecule from one or more other species.

Subject: The term “subject” includes human and non-human animals.Non-human animals include all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dog, cow, chickens,amphibians, and reptiles. Except when noted, the terms “patient” or“subject” are used herein interchangeably.

Target Molecule: The term “target molecule” as used herein refers to anybiological molecule (e.g., protein, carbohydrate, lipid or combinationthereof) expressed on a cell surface or in the extracellular matrix thatcan be specifically bound by a targeting moiety in an IL2 agonist of thedisclosure.

Targeting Moiety: The term “targeting moiety” as used herein refers toany molecule or binding portion (e.g., an immunoglobulin or an antigenbinding fragment) thereof that can bind to a cell surface orextracellular matrix molecule at a site to which an IL2 agonist of thedisclosure is to be localized, for example on tumor cells or onlymphocytes in the tumor microenvironment. The targeting moiety can alsohave a functional activity in addition to localizing an IL2 agonist to aparticular site. For example, a targeting moiety that is an anti-PD1antibody or an antigen binding portion thereof can also exhibitanti-tumor activity or enhance the anti-tumor activity by an IL2 muteinby inhibiting PD1 signaling.

Treat, Treatment, Treating: As used herein, the terms “treat”,“treatment” and “treating” refer to the reduction or amelioration of theprogression, severity and/or duration of a proliferative disorder, orthe amelioration of one or more symptoms (preferably, one or morediscernible symptoms) of a proliferative disorder resulting from theadministration of one or more IL2 agonists of the disclosure. Inspecific embodiments, the terms “treat”, “treatment” and “treating”refer to the amelioration of at least one measurable physical parameterof a proliferative disorder, such as growth of a tumor, not necessarilydiscernible by the patient. In other embodiments the terms “treat”,“treatment” and “treating” refer to the inhibition of the progression ofa proliferative disorder, either physically by, e.g., stabilization of adiscernible symptom, physiologically by, e.g., stabilization of aphysical parameter, or both. In other embodiments the terms “treat”,“treatment” and “treating” refer to the reduction or stabilization oftumor size or cancerous cell count.

Tumor: The term “tumor” is used interchangeably with the term “cancer”herein, e.g., both terms encompass solid and liquid, e.g., diffuse orcirculating, tumors. As used herein, the term “cancer” or “tumor”includes premalignant, as well as malignant cancers and tumors.

Tumor-Associated Antigen: The term “tumor-associated antigen” or “TAA”refers to a molecule (typically a protein, carbohydrate, lipid or somecombination thereof) that is expressed on the surface of a cancer cell,either entirely or as a fragment (e.g., MHC/peptide), and which isuseful for the preferential targeting of a pharmacological agent to thecancer cell. In some embodiments, a TAA is a marker expressed by bothnormal cells and cancer cells, e.g., a lineage marker, e.g., CD19 on Bcells. In some embodiments, a TAA is a cell surface molecule that isoverexpressed in a cancer cell in comparison to a normal cell, forinstance, 1-fold over expression, 2-fold overexpression, 3-foldoverexpression or more in comparison to a normal cell. In someembodiments, a TAA is a cell surface molecule that is inappropriatelysynthesized in the cancer cell, for instance, a molecule that containsdeletions, additions or mutations in comparison to the moleculeexpressed on a normal cell. In some embodiments, a TAA will be expressedexclusively on the cell surface of a cancer cell, entirely or as afragment (e.g., MHC/peptide), and not synthesized or expressed on thesurface of a normal cell. Accordingly, the term “TAA” encompassesantigens that are specific to cancer cells, sometimes known in the artas tumor-specific antigens (“TSAs”).

Universal Light Chain: The term “universal light chain” as used hereinin the context of a targeting moiety refers to a light chain polypeptidecapable of pairing with the heavy chain region of the targeting moietyand also capable of pairing with other heavy chain regions. Universallight chains are also known as “common light chains.”

VH: The term “VH” refers to the variable region of an immunoglobulinheavy chain of an antibody, including the heavy chain of an scFv or aFab.

VL: The term “VL” refers to the variable region of an immunoglobulinlight chain, including the light chain of an scFv or a Fab.

6.2. IL2 Agonists

The present disclosure provides IL2 agonists comprising an IL2 moiety,an optional multimerization moiety, and an optional targeting moiety.

The IL2 agonists of the disclosure can be monomers or multimers, e.g.,dimers (homo dimers or heterodimers) or high order complexes. Forconvenience, IL2 agonists that are homodimers (or higher order multimersof the same polypeptide) are described by their constituent monomers;however, upon recombinant expression of the component monomers in asuitable cell line a homodimeric (or higher order multimer) molecule canbe produced.

In various embodiments, the IL2 agonists are (a) monovalent or bivalentfor the IL2 moiety and/or (b) monovalent or bivalent for a targetingmoiety (if present).

The IL2 agonists of the disclosure and/or the IL2 muteins in the IL2agonists of the disclosure are typically not CD122 directed, e.g., theydo not have amino acid substitutions or other modifications (e.g.,pegylation) in the IL2 moiety that make them preferentially bind toIL-2Rβ as compared to IL-2Rα, relative to wild type IL2. However, incertain embodiments, the IL2 agonists of the disclosure can be CD122directed, for example where the IL2 agonists are pMHC-targeted (e.g., asdescribed in Section 6.4.3) and/or used in combination with CAR Tregtherapy of autoimmune disease, for example as described in Sections6.11.1 and 6.11.1.4. In some embodiments, such IL2 agonists can have upto a 1000 fold reduction in binding affinity for IL-2Rα and up to a 50fold reduction in binding affinity to IL-2Rβ.

The IL2 agonists of the disclosure and/or the IL2 muteins in the IL2agonists of the disclosure can be CD25 directed, e.g., they have one ormore amino acid substitutions or other modifications in the IL2 moietythat make them preferentially bind to the IL-2Rα as compared to IL-2Rβas compared to wild type IL2.

Thus, the IL2 agonists of the disclosure and/or the IL2 muteins in theIL2 agonists of the disclosure can have amino acid modifications thatresult in a reduction in IL-2Rβ and/or IL-2Rα binding affinity.

Overall, the IL2 agonists of the disclosure and/or the IL2 muteins inthe IL2 agonists of the disclosure can have normal or attenuated binding(i.e., reduced affinity) to the intermediate and/or high affinity IL2receptors (e.g., by up to 10-fold, by up to 50-fold, by up to 100-fold,by up to-200 fold, by up to 500-fold, by up to 1,000-fold or by up to5,000-fold). In some embodiments, the binding is attenuated but thepreference for intermediate versus high affinity IL2 receptors issimilar to wild type IL2.

Preferential binding to one receptor vs the other can be evaluated bymeasuring the difference in binding affinity to high affinity receptor(IL-2Rαβγ) expressing cells and intermediate affinity receptor (IL-2Rβγ)expressing cells, and comparing the ratio to the corresponding ratio forwild type IL2.

In certain embodiments, the IL2 agonists of the disclosure have one ormore amino acid substitutions in the IL2 moiety that reduce binding toIL-2Rβ. For example, in some embodiments, the IL2 moiety can have up to100-fold to 1,000-fold attenuated binding to human IL-2Rβ as compared towild-type human IL2.

The IL2 moiety with reduced binding to IL-2Rβ can retain its affinity toIL-2Rα, or have reduced binding to IL-2Rα. For example, in someembodiments, the IL2 moiety can have up to 50-fold, up to 100-fold, upto 500-fold, up to 1,000-fold or up to 5,000 fold attenuated binding tohuman IL2-Ra as compared to wild-type human IL2.

Binding affinity to IL-2Rα, IL-2Rβ, the intermediate affinity receptorand the high affinity receptor can be assayed by surface plasmonresonance (SPR) techniques (analyzed on a BIAcore instrument) (Liljebladet al., 2000, Glyco J 17:323-329).

Exemplary IL2 moieties suitable for use in the IL2 agonists of thedisclosure are described in Section 6.2.

The IL2 agonist can be a fusion protein comprising the IL2 moiety and amultimerization moiety and/or a targeting moiety. Exemplarymultimerization moieties are described in Section 6.5 and include Fcdomains that confer homodimerization or heterodimerization capability tothe IL2 agonist. Free IL2 has very poor pharmacokinetics (a serumhalf-life of <10 min) and, without being bound by theory, it is believedthat the inclusion of a multimerization domain, such as an Fc domain,improves serum stability and the pharmacokinetic profile of an IL2agonist. Thus, the Fc domain can be a dual purpose domain, conferringstabilization properties of a stabilization moiety as described inSection 6.6.

Sometimes, for convenience, the IL2 moiety and optional multimerizationand/or stabilization moiety are referred to herein as an IL2 mutein,although the term “mutein” also encompasses molecules with a targetingmoiety. Exemplary targeting moieties are described in Section 6.4 andinclude an antigen binding domain (e.g., a scFv or Fab) that binds to atumor associated antigen, binds to a tumor microenvironment antigen, orbinds to tumor lymphocytes, as well as a peptide-MHC complex thatrecognizes tumor lymphocytes.

Further, each of the IL2 moiety, multimerization moiety, and targetingmoiety can itself be a fusion protein. For example, the IL2 moiety cancomprise an IL2 or IL2 variant domain and an IL-2Rα domain.

In various embodiments, the IL2 agonist does not comprise (a) a cytokineother than IL2; (b) an anti-IL2 antibody or antibody fragment; (c) ananti-DNA antibody or antibody fragment; (b) a non-binding antibodyvariable domain; any combination two, three or all four of the above.

The IL2 agonist can include one or more linker sequences connecting thevarious components of the molecule, for example the different domainspresent in a fusion protein. Exemplary linkers are described in Section6.7.

In certain aspects, the IL2 agonists of the disclosure, followingadministration to a subject (e.g., a patient with cancer or atumor-bearing mouse), increase ratio CD8+ T cells to Treg cells within atumor.

Below are some illustrative orientations of IL2 muteins and agonists ofthe disclosure, in an N-to-C terminal orientation:

(1) Orientation 1: IL2 moiety—optional linker-multimerization and/orstabilization moiety.

(2) Orientation 2: Multimerization and/or stabilization moiety-optionallinker—IL2 moiety.

(3) Orientation 3: Targeting moiety-optional linker-multimerizationand/or stabilization moiety-optional linker IL-2 moiety.

(4) Orientation 4: A heterodimer comprising two polypeptides, A and B:

-   -   Polypeptide A: IL2 moiety—optional linker-multimerization        moiety; and    -   Polypeptide B: Targeting moiety-optional linker-multimerization        moiety.

In the IL2 agonists of the disclosure, when the targeting moiety is anantigen binding domain (“ABD”) of an antibody, each IL2 molecule can becomposed of two polypeptide chains, one polypeptide chain bearing theheavy chain variable region and the other polypeptide chain bearing thelight chain variable region. Thus, in Orientation 3 and Orientation 4,the targeting moiety itself can comprise heavy and light chain variabledomains on separate polypeptide chains. For example, with respect anOrientation 4 IL2 agonist, Polypeptide B can be composed of twopolypeptide chains, B-1 and B-2. Polypeptide chain B-1 can contain theheavy chain variable domain of a targeting moiety-optionallinker-multimerization moiety, and polypeptide chain B-2 can comprisethe light chain variable domain of a targeting moiety. The PolypeptideA-Polypeptide B heterodimer can be associated through the pairing of Fcheterodimerization variants as described in Section 6.5.1.2.

Alternatively, an scFv can be used in which the heavy and light chainvariable regions are fused to one another in a single polypeptide.

An exemplary embodiment of Orientation 1 IL2 agonist comprises, in an N-to C-terminal orientation:

-   -   a) IL2 moiety comprising:        -   i) IL2 or IL2 variant (e.g., IL2 N88D) domain, with or            without a substitution at C125 that reduces aggregation            (e.g., C125S or C125A or C125V);        -   ii) Linker (e.g., as described in Section 6.7); and        -   iii) IL2 binding portion of IL-2Rα;    -   b) Linker (e.g., as described in Section 6.7); and    -   c) Fc domain (e.g., IgG1 or IgG4, with or without substitutions        that reduce glycosylation and/or effector function as described        in Section 6.5.1 and subsections thereof).

The IL2 agonists referred to as IL2M0 and IL2M2 in Section 7 areparticular embodiments of Orientation 1 IL2 agonists.

An exemplary embodiment of an Orientation 2 IL2 agonist comprises, in anN- to C-terminal orientation:

-   -   a) Fc domain (e.g., IgG1 or IgG4, with or without substitutions        that reduce glycosylation and/or effector function as described        in Section 6.5.1 and subsections thereof);    -   b) Linker (e.g., as described in Section 6.7); and    -   c) IL2 or IL2 variant (e.g., IL2 N88D) domain, with or without a        substitution at C125 that reduces aggregation (e.g., C125S or        C125A or C125V).

The IL2 agonist referred to as IL2M4 and IL2M5 are particularembodiments of these Orientation 2 IL2 agonists.

Another exemplary embodiment of an Orientation 2 IL2 agonist comprises,in an N- to C-terminal orientation:

-   -   a) Fc domain (e.g., IgG1 or IgG4, with or without substitutions        that reduce glycosylation and/or effector function as described        in Section 6.5.1 and subsections thereof);    -   b) Linker (e.g., as described in Section 6.7); and    -   c) IL2 moiety comprising:        -   i) IL2 or IL2 variant (e.g., IL2 N88D) domain, with or            without a substitution at C125 that reduces aggregation            (e.g., C125S or C125A or C125V);        -   ii) Linker (e.g., as described in Section 6.7); and        -   iii) IL2 binding portion of IL-2Rα.

The IL2 agonist referred to as IL2M3 in Section 7 is a particularembodiment of this Orientation 2 IL2 agonist.

An exemplary embodiment of an Orientation 3 IL2 agonist comprises, in anN- to C-terminal orientation:

-   -   a) scFv or a heavy chain variable region of a Fab (associated        with a corresponding light chain variable region on a separate        polypeptide) (e.g., as described in Section 6.4.2 and        subsections thereof);    -   b) Linker (e.g., as described in Section 6.7);    -   c) Fc domain (e.g., IgG1 or IgG4, with or without substitutions        that reduce glycosylation and/or effector function as described        in Section 6.5.1 and subsections thereof);    -   d) Linker (e.g., as described in Section 6.7); and    -   e) An IL2 moiety comprising:        -   i) IL2 or IL2 variant (e.g., IL2 N88D) domain, with or            without a substitution at C125 that reduces aggregation            (e.g., C125S, C125A or C125V);        -   ii) Linker (e.g., as described in Section 6.7); and        -   iii) IL2 binding portion of IL-2Rα (e.g., as described in            Section 6.3).

The T1 targeted version of the IL2 agonist referred to as IL2M3 inSection 7 is a particular embodiment of this Orientation 3 IL2 agonist.

Another exemplary embodiment of an Orientation 3 IL2 agonist comprises,in an N- to C-terminal orientation:

-   -   a) Peptide-MHC complex (e.g., as described in Section 6.4.3)        comprising:        -   i) MHC peptide;        -   ii) Linker (e.g., as described in Section 6.7 or subsections            thereof, for example in Section 6.7.1);        -   iii) Optional β2-microglobulin (β2m);        -   iv) Optional linker (e.g., as described in Section 6.7 or            subsections thereof, for example in Section 6.7.1); and        -   v) MHC;    -   b) Optional linker (e.g., as described in Section 6.7);    -   c) Fc domain (e.g., IgG1 or IgG4, with or without substitutions        that reduce glycosylation and/or effector function as described        in Section 6.5.1 and subsections thereof);    -   d) Linker (e.g., as described in Section 6.7);    -   e) IL2 moiety comprising:        -   i) IL2 or IL2 variant (e.g., IL2 N88D) domain, with or            without a substitution at C125 that reduces aggregation            (e.g., C125S, C125A or C125V);        -   ii) Linker (e.g., as described in Section 6.7); and        -   iii) IL2 binding portion of IL-2Rα (e.g., as described in            Section 6.3).

The T2 and T3 targeted versions of the IL2 agonist referred to as IL2M3in Section 7 are particular embodiments of this Orientation 3 IL2agonist.

In an exemplary embodiment, an Orientation 4 IL2 agonist comprises twopolypeptides, A and B, comprising, in an N- to C-terminal orientation:

In certain aspects, an Orientation 4 IL2 agonist comprises twopolypeptides, A and B, comprising, in an N- to C-terminal orientation:

-   -   Polypeptide A:    -   a) Targeting moiety, e.g., peptide-MHC complex (e.g., as        described in Section 6.4.3), Fab domain (e.g., as described in        Section 6.4.2.2) (e.g., a heavy chain of a Fab on polypeptide A        associated with a third polypeptide, polypeptide C, comprising        the light chain of the Fab), or scFv domain (e.g., as described        in Section 6.4.2.1);    -   b) Optional linker (e.g., as described in Section 6.7); and    -   c) A first Fc domain.    -   Polypeptide B:    -   d) IL2 moiety comprising an IL2 or IL2 variant domain (e.g., an        IL2 domain with the substitutions H16A, F42A, also referred to        as IL2(2m)), with or without a substitution at C125 that reduces        aggregation (e.g., C125S, C125A or C125V);    -   e) An optional linker (e.g., as described in Section 6.7); and    -   f) A second Fc domain that is not identical to, but can        heterodimerize with, the first Fc domain (e.g., as described in        Section 6.5.1.2).

In some embodiments, the IL2 agonist is a heterodimer that is monovalentfor the targeting moiety and monovalent for the IL2 moiety.

A particular embodiment of this Orientation 4 IL2 agonist comprises:

-   -   Polypeptide A:    -   a) Peptide-MHC complex (e.g., as described in Section 6.4.3)        comprising:        -   i) MHC peptide;        -   ii) Linker (e.g., as described in Section 6.7);        -   iii) Optional β2-microglobulin (β2m);        -   iv) Optional linker (e.g., as described in Section 6.7); and        -   v) MHC;    -   b) Optional linker (e.g., as described in Section 6.7); and    -   c) A first Fc domain.    -   Polypeptide B:    -   d) IL2 moiety comprising an IL2 or IL2 variant (e.g., IL2 H16A,        F42A, also referred to as IL2(2m)) domain, with or without a        substitution at C125 that reduces aggregation (e.g., C125S,        C125A or C125V);    -   e) An optional linker (e.g., as described in Section 6.7); and    -   f) A second Fc domain that is not identical to, but can        heterodimerize with, the first Fc domain (e.g., as described in        Section 6.5.1.2).

The first and second Fc domains can be e.g., IgG1 or IgG4 Fc domains,with or without substitutions that reduce glycosylation and/or effectorfunction as described in Section 6.5.1 and subsections thereof.

An exemplary Orientation 4 IL2 agonist is illustrated in FIG. 22A.2.

The inclusion of a β2 microglobulin in a peptide-MHC complex, forexample in the exemplary Orientation 3 and Orientation 4 agonistsdescribed above, is believed to stabilize human cell surface MHC Imolecules and facilitate their loading with exogenous peptides. See,e.g., Shields et al., 1998, J Biol Chem. 273(43):28010-8; Obermann etal., 2007, Immunology 122(1):90-7.

In certain aspects, the IL2 agonist comprises an IL2 mutein which hasthe configuration of IL2M0, IL2M1, IL2M2, IL2M3, IL2M4, IL2M5, IL2M6, orIL2M7, with an optional targeting moiety and an optional linker, e.g.,at the N-terminus. In some embodiments, the IL2 agonist comprises an IL2mutein which has the amino acid sequence of IL2M0, IL2M1, IL2M2, IL2M3,IL2M4, IL2M5, IL2M6, or IL2M7, with an optional targeting moiety and anoptional linker, e.g., at the N-terminus.

The IL2 agonists of the disclosure generally have improved therapeuticindices. In certain aspects, the therapeutic index can be measured as aratio of the dose causing toxicity (e.g., loss of body weight) in tumorbearing mice vs. the minimum effective anti-tumor dose, of the IL2agonist as a whole or of a non-targeting component thereof (e.g., amolecule comprising the IL2 moiety and the multimerization moiety, forconvenience referred to herein the “IL2 mutein” or “IL2M” component) asexemplified in Section 7. The data in Section 7 shows the followingtherapeutic indices:

IL2M0 IL2M1 IL15M1 Efficacious dose  >5 μg >75 μg >20 μg Toxic dose >30μg >40 μg >10 μg Therapeutic index 6 0.53 0.5

In certain aspects, an IL2 agonist of the disclosure, or of an IL2Mcomponent thereof, has a therapeutic index of greater than 1, andpreferably greater than 2, and even more preferably greater than 10. Inparticular embodiments, the therapeutic index is about 10, about 20,about 100, or about 200.

Further details of the components of the IL2 agonists of the disclosureare presented below.

6.3. The IL2 Moiety

The IL2 moiety of the IL2 antagonists of the disclosure comprises a wildtype or variant IL2 domain, which is optionally fused to an IL2 bindingdomain of IL-2Rα, optionally via a linker (e.g., as described in Section6.7). When present, the IL2 binding domain of IL-2Rα can be N-terminalor C-terminal to the wild type or variant IL2 domain.

In eukaryotic cells human IL2 is synthesized as a precursor polypeptideof 153 amino acids, from which 20 amino acids are removed to generatemature secreted IL2 (Taniguchi et al., 1983, Nature 302(5906):305-10).Mature human IL2 has the following amino acid sequence:

-   -   APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE        EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRW        ITFCQSIISTLT (SEQ ID NO:2)

The IL2 moieties of the disclosure are typically not CD122 directed,e.g., they do not have amino acid substitutions in the IL2 domain thatmake them preferentially bind to IL-2Rβ as compared to IL-2Rα.

The IL2 moieties of the disclosure can be CD25 directed, e.g., they haveone or more amino acid substitutions in the IL2 domain that make thempreferentially bind to IL-2Rα as compared to IL-2Rβ.

In certain embodiments, the IL2 agonists of the disclosure have one ormore amino acid substitutions in the IL2 moiety that reduce binding toIL-2Rβ. For example, in some embodiments, the IL2 moiety can have up to50-fold (and in some embodiments up to 100-fold) to 1,000-foldattenuated binding to human IL-2Rβ as compared to wild-type human IL2.

The IL2 moiety with reduced binding to IL-2Rβ can retain its affinity toIL-2Rα, or have reduced binding to IL-2Rα. For example, in someembodiments, the IL2 moiety can have up to 50-fold attenuated binding tohuman IL2-Ra as compared to wild-type human IL2.

Other characteristics of useful IL2 variants may include the ability toinduce proliferation of IL-2Rα-bearing CD8+ T cells in tumors, theability to induce IL2 signaling in IL-2Rα-bearing CD8+ T cells intumors, and an improved therapeutic index.

In one embodiment, the IL2 moiety comprises one or more amino acidsubstitutions that reduce affinity to IL-2Rβ and preserve affinity toIL2-Ra. An exemplary amino acid substitution is N88D. Other amino acidsubstitutions that reduce or abolish the affinity of IL2 to IL-2Rβ areD20T, N88R, N88D or Q126D (see e.g., US Patent Publication No. US2007/0036752).

In one embodiment, the IL2 moiety comprises one or more amino acidsubstitutions that reduce affinity to IL2-Rα and preserve, or reducesaffinity to a lesser degree, to IL-2Rβ, resulting in CD122 directed IL2moieties. Such IL2 moieties are particularly useful to be incorporatedinto IL2 agonists with peptide-MHC targeting moieties and/or used asadjunct therapy for CAR Treg therapy, as disclosed in Section 6.11.1.4.Exemplary CD122 directed IL2 moieties are those comprising both H16A andF42A substitutions, as exemplified in IL2M6 and IL2M7. Accordingly, insome embodiments, the IL2 moiety comprises the amino acid sequence ofhuman IL2 with H16A and F42A substitutions, as shown below:

(SEQ ID NO: 124) SAPTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

In certain embodiments, the IL2 moiety comprises an amino acidsubstitution which eliminates the O-glycosylation site of IL2 at aposition corresponding to residue 3 of human IL2. Exemplary amino acidsubstitutions at T3 are T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P.In a specific embodiment, the substitution is T3A.

The IL2 moiety is preferably essentially a full-length IL2 molecule,e.g., a human IL2 molecule. In certain embodiments the IL2 moiety is ahuman IL-2 molecule.

C125 can be substituted with S, V, or A to reduce protein aggregation,as described in U.S. Pat. No. 4,518,584.

As described therein, one may also delete the N-terminal alanine residueof IL2, resulting in des-A1 IL2.

Further, the IL2 moiety may include a substitution of methionine 104with a neutral amino acid such as alanine, as described in U.S. Pat. No.5,206,344.

Accordingly, the IL2 moieties of the disclosure can have amino aciddeletions and/or substitutions selected from des-A1 M104A IL2, des-A1M104A C125S IL2, M104A IL2, M104A C125A IL2, des-A1 M104A C125A IL2, orM104A C125S IL2, in addition to other variations alter the binding ofIL2 to its receptor. These and other mutants may be found in U.S. Pat.No. 5,116,943 and in Weiger et al., 1989, Eur J Biochem 180:295-300.

In various aspects, any of the foregoing IL2 moieties comprises an aminoacid sequence having at least about 90%, at least about 91%, at leastabout 92%, about at least 93%, at least about 94%, at eat least about95%, at least about 96%, at least about 97%, at least about 98%, atleast about 99% or 100% sequence identity to mature human IL2.

The IL2 moieties of the disclosure can further include an IL2 bindingdomain of IL-2Rα (referred to as the “IL2-Rα domain” for convenience),e.g., the extracellular domain of an IL-2Rα, fused at the C-terminus orthe N-terminus of IL2, optionally via a linker as described in Section6.7. The sequence of the mature human IL-2Rα extracellular domain(corresponding to amino acids 22-272 of human IL-Rα) is:

(SEQ ID NO: 3) Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro HisAla Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly ThrMet Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg ArgIle Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr GlyAsn Ser Ser His Ser Ser Trp Asp Asn Gln Cys GlnCys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys GlnVal Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg LysThr Thr Glu Met Gln Ser Pro Met Gln Pro Val AspGln Ala Ser Leu Pro Gly His Cys Arg Glu Pro ProPro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr HisPhe Val Val Gly Gln Met Val Tyr Tyr Gln Cys ValGln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala GluSer Val Cys Lys Met Thr His Gly Lys Thr Arg TrpThr Gln Pro Gln Leu Ile Cys Thr Gly Glu Met GluThr Ser Gln Phe Pro Gly Glu Glu Lys Pro Gln AlaSer Pro Glu Gly Arg Pro Glu Ser Glu Thr Ser CysLeu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr GluMet Ala Ala Thr Met Glu Thr Ser Ile Phe Thr ThrGlu Tyr Gln Val Ala Val Ala Gly Cys Val Phe LeuLeu Ile Ser Val Leu Leu Leu Ser Gly Leu Thr TrpGln Arg Arg Gln Arg Lys Ser Arg Arg Thr Ile

The sequence of an IL2 binding portion of the human IL-2Rα extracellulardomain (comprising the two “sushi” domains, which corresponds to aminoacids 22-186 of human IL-2Rα) is:

(SEQ ID NO: 4) Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro HisAla Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly ThrMet Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg ArgIle Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr GlyAsn Ser Ser His Ser Ser Trp Asp Asn Gln Cys GlnCys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys GlnVal Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg LysThr Thr Glu Met Gln Ser Pro Met Gln Pro Val AspGln Ala Ser Leu Pro Gly His Cys Arg Glu Pro ProPro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr HisPhe Val Val Gly Gln Met Val Tyr Tyr Gln Cys ValGln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala GluSer Val Cys Lys Met Thr His Gly Lys Thr Arg TrpThr Gln Pro Gln Leu Ile Cys Thr Gly

The sequence of an alternative IL2 binding portion of the human IL-2Rαextracellular domain, which corresponds to amino acids 22-240 of humanIL-2Rα, is:

(SEQ ID NO: 5) Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro HisAla Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly ThrMet Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg ArgIle Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr GlyAsn Ser Ser His Ser Ser Trp Asp Asn Gln Cys GlnCys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys GlnVal Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg LysThr Thr Glu Met Gln Ser Pro Met Gln Pro Val AspGln Ala Ser Leu Pro Gly His Cys Arg Glu Pro ProPro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr HisPhe Val Val Gly Gln Met Val Tyr Tyr Gln Cys ValGln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala GluSer Val Cys Lys Met Thr His Gly Lys Thr Arg TrpThr Gln Pro Gln Leu Ile Cys Thr Gly Glu Met GluThr Ser Gln Phe Pro Gly Glu Glu Lys Pro Gln AlaSer Pro Glu Gly Arg Pro Glu Ser Glu Thr Ser CysLeu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr GluMet Ala Ala Thr Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln

The IL2-Rα domain or the IL2 binding portion of the IL-2Rα extracellulardomain preferably has an amino acid sequence with at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99% or 100% sequence identity to any of thesequences above, i.e., any one of amino acids 22-186 of IL-2Rα, aminoacids 22-240 of IL-2Rα, or amino acids 22-272 of IL-2Rα, or any IL2binding portion thereof.

In certain aspects, the IL2-Rα domain or the IL2 binding portion cancomprise or consist of an amino acid sequence having at least about 90%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99% or 100% sequence identity to an IL2binding portion of human IL-2Rα, optionally wherein the binding portionhas an amino acid sequence of (a) at least 160 amino acids, at least 161amino acids, at least 162 amino acids, at least 164 amino acids or atleast 165 amino acids and/or (b) up to 251, up to 240, up to 230, up to220, up to 210, up to 200, up to 190, up to 180 or up to 170 amino acidsof the extracellular domain of human IL2-Rα. In particular embodiments,the portion of human IL-2Rα is bounded by any one of (a) and (b) in thepreceding sentence, e.g., at least 160 and up to 180 amino acids fromhuman IL-2Rα, at least 162 and up to 200 amino acids from human IL-2Rα,at least 160 and up to 220 amino acids from human IL-2Rα, at least 164and up to 190 amino acids from human IL-2Rα, and so on and so forth.

In some embodiments, the IL2-Rα domain or the IL2 binding portioncomprises or consists of an amino acid sequence having at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99% or 100% sequence identity to aminoacids 22-186, with or without an additional up to 5 amino acids, up to10 amino acids, up to 15 amino acids, up to 20 amino acids, up to 30amino acids, or up to 40 amino acids C-terminal to amino acid residue186, of IL2-Rα.

As illustrated in FIGS. 3A-3B, the fusion of IL2 to the C-terminus ofthe IL2 binding portion of IL-2Rα, with an N-terminal Fc domain, resultsin IL2 agonists that do not form higher order structures as with an IL2agonist comprising, in an N-to-C direction, IL2-IL-2Rα extracellulardomain-Fc. Accordingly, the IL-2Rα-containing IL2 muteins of thedisclosure preferably have the IL-2Rα extracellular domain at theN-terminus of IL2. An exemplary mutein with this orientation is IL2M3.

In certain embodiments, the IL2-Rα domain or the IL-2Rα extracellulardomain has at least one fewer O-glycosylation and/or N-glycosylationcompared to the extracellular domain of native IL-2Rα, for example by asubstitution at one or more of amino acid N49, amino acid N68, aminoacid T74, amino acid T85, amino acid T197, amino acid T203, amino acidT208, and amino acid T216. In some embodiments, the one or moresubstitutions are from asparagine to an amino acid selected from thegroup consisting of alanine, threonine, serine, arginine, aspartic acid,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, tryptophan, tyrosine, andvaline. In some embodiments, the one or more substitutions are fromthreonine to an amino acid selected from the group consisting ofalanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, tryptophan, tyrosine, andvaline. In some embodiments, the one or more substitutions are at aminoacid S50 (e.g., 550P), amino acid S51 (e.g., S51R, S51N, S51D, S51C,S51Q, S51E, S51G, S51H, S51I, S51L, S51K, S51M, S51F, S51P, S51W, S51Y,or S51V), amino acid T69 (e.g., T69P), amino acid T70 (e.g., T70R, T70N,T70D, T70C, T70Q, T70E, T70G, T70H, T70I, T70L, T70K, T70M, T70F, T70P,T70W, T70Y, or T70V, amino acid C192 (e.g., C192R, C192N, C192D, C192Q,C192E, C192G, C192H, C1921, C192L, C192K, C192M, C192F, C192P, C192W,C192Y, or C192V), or any combination thereof.

6.4. The Targeting Moiety

The incorporation of targeting moieties in the IL2 agonists of thedisclosure permits the delivery of high concentrations of IL2 into thetumor microenvironment or to tumor reactive lymphocytes (including CARTlymphocytes) with a concomitant reduction of systemic exposure,resulting in fewer side effects than obtained with wild type IL2.

Suitable targeting moiety formats are described in Section 6.4.2. Thetargeting moiety is preferably an antigen binding moiety, for example anantibody or an antigen-binding portion of an antibody, e.g., an scFv, asdescribed in Section 6.4.2.1, or a Fab, as described in Section 6.4.2.2.

The antibodies and antigen-binding portions generally bind to specificantigenic determinants and are able to direct the IL2 agonist to atarget site, for example to a specific type of tumor cell or tumorstroma that bears the antigenic determinant. Exemplary target moleculesrecognized by the targeting moieties of the disclosure are described inSection 6.4.1.

In other embodiments, the targeting moiety is a peptide-MHC complex, asdescribed in 6.4.3, e.g., a peptide-MHC complex that is recognized bytumor lymphocytes.

In some embodiments, the targeting moiety is in the form of an Fc fusionprotein, for example as exemplified in the structures referred to in theExamples as T7 and T8. The Fc portion of the targeting moietypolypeptide can be used to multimerize with an Fc domain in a separatepolypeptide chain containing an IL2 moiety.

6.4.1. Target Molecules

The target molecules recognized by the targeting moieties of the IL2agonists of the disclosure are generally found, for example, on thesurfaces of activated T cells, on the surfaces of tumor cells, on thesurfaces of virus-infected cells, on the surfaces of other diseasedcells, free in blood serum, in the extracellular matrix (ECM), or immunecells present in the target site, e.g., tumor reactive lymphocytes.Where the immune cells are exogenously administered (e.g., chimericantigen receptor (“CAR”) expressing T cells), the targeting moiety canrecognize the chimeric antigen receptor (CAR) or another molecule foundon the surface of the CAR T cells. In various embodiments, the CAR iscomprises CDRs or VH and VL sequences (e.g., in the format of an scFv)that specifically recognize a TAA or a pMHC complex.

Exemplary target molecules are Fibroblast Activation Protein (FAP), theA1 domain of Tenascin-C (TNC A1), the A2 domain of Tenascin-C (TNC A2),the Extra Domain B of Fibronectin (EDB), the Melanoma-associatedChondroitin Sulfate Proteoglycan (MCSP), MART-1/Melan-A, gp100,Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein(ADAbp), cyclophilin b, colorectal associated antigen(CRC)-C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenicepitopes CAP-1 and CAP-2, etv6, aml1, Prostate Specific Antigen (PSA)and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specificmembrane antigen (PSMA), T-cell receptor/CD3-zeta chain, MAGE-family oftumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5,MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12,MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1,MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05), GAGE-family of tumor antigens(e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8,GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53,MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein, E-cadherin,α-catenin, β-catenin and γ-catenin, p120ctn, gp100 Pmel117, PRAME,NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin,Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viralproducts such as human papilloma virus proteins, Smad family of tumorantigens, Imp-1, PIA, EBV-encoded nuclear antigen (EBNA)-1, brainglycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5,SCP-1 and CT-7, c-erbB-2, Her2, EGFR, IGF-1R, CD2 (T-cell surfaceantigen), CD3 (heteromultimer associated with the TCR), CD22 (B-cellreceptor), CD23 (low affinity IgE receptor), CD30 (cytokine receptor),CD33 (myeloid cell surface antigen), CD40 (tumor necrosis factorreceptor), IL-6R-(IL6 receptor), CD20, MCSP, PDGFβR (β-platelet-derivedgrowth factor receptor), ErbB2 epithelial cell adhesion molecule(EpCAM), EGFR variant III (EGFRvIII), CD19, disialoganglioside GD2,ductal-epithelial mucine, gp36, TAG-72, glioma-associated antigen,β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactiveAFP, thyroglobulin, MN-CA IX, human telomerase reverse transcriptase,RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF,prostase, prostase specific antigen (PSA), PAP, LAGA-1a, p53, prostein,PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1(PCTA-1), ELF2M, neutrophil elastase, ephrin B2, insulin growth factor(IGF1)-I, IGF-II, IGFI receptor, 5T4, ROR1, Nkp30, NKG2D, tumor stromalantigens, the extra domain A (EDA) and extra domain B (EDB) offibronectin and the A1 domain of tenascin-C (TnC A1).

Non-limiting examples of viral antigens include an EBV antigen (e.g.,Epstein-Barr virus LMP-1), a hepatitis C virus antigen (e.g., hepatitisC virus E2 glycoprotein), an HIV antigen (e.g., HIV gp160, and HIVgp120); a CMV antigen; a HPV-specific antigen, or an influenza virusantigen (e.g., influenza virus hemagglutinin).

Non-limiting examples of ECM antigens include syndecan, heparanase,integrins, osteopontin, link, cadherins, laminin, laminin type EGF,lectin, fibronectin, notch, tenascin, collagen and matrixin.

Other target molecules are cell surface molecules of tumor or virallymphocytes, for example T-cell co-stimulatory proteins such as CD27,CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, andB7-H3.

In particular embodiments, the target molecules are checkpointinhibitors, for example CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA,HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2.In particular embodiments, the target molecule is PD1. In otherembodiments, the target molecule is LAG3.

6.4.2. Targeting Moiety Format

In certain aspects, the targeting moiety can be any type of antibody orfragment thereof that retains specific binding to an antigenicdeterminant. In one embodiment the antigen binding moiety is afull-length antibody. In one embodiment the antigen binding moiety is animmunoglobulin molecule, particularly an IgG class immunoglobulinmolecule, more particularly an IgG₁ or IgG₄ immunoglobulin molecule.Antibody fragments include, but are not limited to, VH (or V_(H))fragments, VL (or V_(L)) fragments, Fab fragments, F(ab′)₂ fragments,scFv fragments, Fv fragments, minibodies, diabodies, triabodies, andtetrabodies.

6.4.2.1. scFv

Single chain Fv or “scFv” antibody fragments comprise the VH and VLdomains of an antibody in a single polypeptide chain, are capable ofbeing expressed as a single chain polypeptide, and retain thespecificity of the intact antibodies from which they are derived.Generally, the scFv polypeptide further comprises a polypeptide linkerbetween the VH and VL domain that enables the scFv to form the desiredstructure for target binding. Examples of linkers suitable forconnecting the VH and VL chains of an scFv are the linkers identified inSection 6.4.3.

Unless specified, as used herein an scFv may have the VL and VH variableregions in either order, e.g., with respect to the N-terminal andC-terminal ends of the polypeptide, the scFv may comprise VL-linker-VHor may comprise VH-linker-VL.

The scFv can comprise VH and VL sequences from any suitable species,such as murine, human or humanized VH and VL sequences.

To create an scFv-encoding nucleic acid, the VH and VL-encoding DNAfragments are operably linked to another fragment encoding a linker,e.g., encoding any of the linkers described in Section 6.4.3 (typicallya repeat of a sequence containing the amino acids glycine and serine,such as the amino acid sequence (Gly4˜Ser)₃ (SEQ ID NO:6), such that theVH and VL sequences can be expressed as a contiguous single-chainprotein, with the VL and VH regions joined by the flexible linker (see,e.g., Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc.Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature348:552-554).

6.4.2.2. Fab

Fab domains were traditionally produced from by proteolytic cleavage ofimmunoglobulin molecules using enzymes such as papain. In the IL2agonists of the disclosure, the Fab domains are typically recombinantlyexpressed as part of the IL2 agonist.

The Fab domains can comprise constant domain and variable regionsequences from any suitable species, and thus can be murine, chimeric,human or humanized.

Fab domains typically comprise a CH1 domain attached to a VH domainwhich pairs with a CL domain attached to a VL domain. In a wild-typeimmunoglobulin, the VH domain is paired with the VL domain to constitutethe Fv region, and the CH1 domain is paired with the CL domain tofurther stabilize the binding module. A disulfide bond between the twoconstant domains can further stabilize the Fab domain.

For the IL2 agonists of the disclosure, particularly when the lightchain is nota common or universal light chain, it is advantageous to useFab heterodimerization strategies to permit the correct association ofFab domains belonging to the same ABD and minimize aberrant pairing ofFab domains belonging to different ABDs. For example, the Fabheterodimerization strategies shown in Table 1 below can be used:

TABLE 1 Fab Heterodimerization Strategies STRATEGY VH CH1 VL CLREFERENCE CrossMabCH1- WT CL domain WT CH1 domain Schaefer et al., CL2011, Cancer Cell 2011; 20: 472-86; PMID: 22014573. orthogonal Fab 39K,H172A, 1R, 38D, L135Y, Lewis et al., 2014, VHVRD1CH1CRD2 - 62E F174G(36F) S176W Nat Biotechnol 32: VLVRD1CλCRD2 191-8 orthogonal Fab 39Y WT38R WT Lewis et al., 2014, VHVRD2CH1wt - Nat Biotechnol 32: VLVRD2Cλwt191-8 TCR CαCβ 39K TCR Cα 38D TCR Cβ Wu et al., 2015, MAbs 7: 364-76 CR3WT T192E WT N137K, Golay at al., 2016, S114A J Immunol 196: 3199- 211.MUT4 WT L143Q, WT V133T, Golay at al., 2016, S188V S176V J Immunol 196:3199- 211. DuetMab WT F126C WT S121C Mazor et al., 2015, MAbs 7: 377-89;Mazor et al., 2015, MAbs 7: 461-669. Domain WT CH3 + knob WT CH3 + holeWozniak-Knopp et al., exchanged or hole or knob 2018, PLoSONE13(4):mutation mutation e0195442

Accordingly, in certain embodiments, correct association between the twopolypeptides of a Fab is promoted by exchanging the VL and VH domains ofthe Fab for each other or exchanging the CH1 and CL domains for eachother, e.g., as described in WO 2009/080251.

Correct Fab pairing can also be promoted by introducing one or moreamino acid modifications in the CH1 domain and one or more amino acidmodifications in the CL domain of the Fab and/or one or more amino acidmodifications in the VH domain and one or more amino acid modificationsin the VL domain. The amino acids that are modified are typically partof the VH:VL and CH1:CL interface such that the Fab componentspreferentially pair with each other rather than with components of otherFabs.

In one embodiment, the one or more amino acid modifications are limitedto the conserved framework residues of the variable (VH, VL) andconstant (CH1, CL) domains as indicated by the Kabat numbering ofresidues. Almagro, 2008, Frontiers In Bioscience 13:1619-1633 provides adefinition of the framework residues on the basis of Kabat, Chothia, andIMGT numbering schemes.

In one embodiment, the modifications introduced in the VH and CH1 and/orVL and CL domains are complementary to each other. Complementarity atthe heavy and light chain interface can be achieved on the basis ofsteric and hydrophobic contacts, electrostatic/charge interactions or acombination of the variety of interactions. The complementarity betweenprotein surfaces is broadly described in the literature in terms of lockand key fit, knob into hole, protrusion and cavity, donor and acceptoretc., all implying the nature of structural and chemical match betweenthe two interacting surfaces.

In one embodiment, the one or more introduced modifications introduce anew hydrogen bond across the interface of the Fab components. In oneembodiment, the one or more introduced modifications introduce a newsalt bridge across the interface of the Fab components. Exemplarysubstitutions are described in WO 2014/150973 and WO 2014/082179, thecontents of which are hereby incorporated by reference.

In some embodiments, the Fab domain comprises a 192E substitution in theCH1 domain and 114A and 137K substitutions in the CL domain, whichintroduces a salt-bridge between the CH1 and CL domains (see, e.g.,Golay et al., 2016, J Immunol 196:3199-211).

In some embodiments, the Fab domain comprises a 143Q and 188Vsubstitutions in the CH1 domain and 113T and 176V substitutions in theCL domain, which serves to swap hydrophobic and polar regions of contactbetween the CH1 and CL domain (see, e.g., Golay et al., 2016, J Immunol196:3199-211).

In some embodiments, the Fab domain can comprise modifications in someor all of the VH, CH1, VL, CL domains to introduce orthogonal Fabinterfaces which promote correct assembly of Fab domains (Lewis et al.,2014 Nature Biotechnology 32:191-198). In an embodiment, 39K, 62Emodifications are introduced in the VH domain, H172A, F174Gmodifications are introduced in the CH1 domain, 1 R, 38D, (36F)modifications are introduced in the VL domain, and L135Y, S176Wmodifications are introduced in the CL domain. In another embodiment, a39Y modification is introduced in the VH domain and a 38R modificationis introduced in the VL domain.

Fab domains can also be modified to replace the native CH1:CL disulfidebond with an engineered disulfide bond, thereby increasing theefficiency of Fab component pairing. For example, an engineereddisulfide bond can be introduced by introducing a 126C in the CH1 domainand a 121 C in the CL domain (see, e.g., Mazor et al., 2015, MAbs7:377-89).

Fab domains can also be modified by replacing the CH1 domain and CLdomain with alternative domains that promote correct assembly. Forexample, Wu et al., 2015, MAbs 7:364-76, describes substituting the CH1domain with the constant domain of the a T cell receptor andsubstituting the CL domain with the b domain of the T cell receptor, andpairing these domain replacements with an additional charge-chargeinteraction between the VL and VH domains by introducing a 38Dmodification in the VL domain and a 39K modification in the VH domain.

In lieu of, or in addition to, the use of Fab heterodimerizationstrategies to promote correct VH-VL pairings, the VL of common lightchain (also referred to as a universal light chain) can be used for eachFab VL region of an IL2 agonist of the disclosure. In variousembodiments, employing a common light chain as described herein reducesthe number of inappropriate species of IL2 agonists as compared toemploying original cognate VLs. In various embodiments, the VL domainsof the IL2 agonists are identified from monospecific antibodiescomprising a common light chain. In various embodiments, the VH regionsof the IL2 agonists comprise human heavy chain variable gene segmentsthat are rearranged in vivo within mouse B cells that have beenpreviously engineered to express a limited human light chain repertoire,or a single human light chain, cognate with human heavy chains and, inresponse to exposure with an antigen of interest, generate an antibodyrepertoire containing a plurality of human VHs that are cognate with oneor one of two possible human VLs, wherein the antibody repertoirespecific for the antigen of interest. Common light chains are thosederived from a rearranged human Vκ1-39Jκ5 sequence or a rearranged humanVκ3-20Jκ1 sequence, and include somatically mutated (e.g., affinitymatured) versions. See, for example, U.S. Pat. No. 10,412,940.

6.4.3. MHC-Peptide Fusions

The targeting moiety of an IL2 agonist of the disclosure can be apeptide-MHC complex (a “pMHC compex”), e.g., a peptide complexed with anMHC class I domain or a peptide complexed with an MHC class II domain,optionally with a β2 microglobulin domain.

Naturally-occurring MHCs are encoded by a cluster of genes on humanchromosome 6. MHCs include, but are not limited to, HLA specificitiessuch as A (e.g., A1-A74), B (e.g., B1-B77), C (e.g., C1-C11), D (e.g.,D1-D26), DR (e.g., DR1-DR8), DQ (e.g., DQ1-DQ9) and DP (e.g., DP1-DP6).HLA specificities include A1, A2, A3, AII, A23, A24, A28, A30, A33, B7,B8, B35, B44, B53, B60, B62, DR1, DR2, DR3, DR4, DR7, DR8, and DR11.

Naturally occurring MHC class I molecules bind peptides derived fromproteolytically degraded proteins. Small peptides obtained accordinglyare transported into the endoplasmic reticulum where they associate withnascent MHC class I molecules before being routed through the Golgiapparatus and displayed on the cell surface for recognition by cytotoxicT lymphocytes, as illustrated in FIG. 3A.

Naturally occurring MHC class I molecules consist of an α (heavy) chainassociated with β2 microglobulin. The heavy chain consists of subunitsα1-α3. The β2 microglobulin protein and α3 subunit of the heavy chainare associated. In certain embodiments, β2 microglobulin and α3 subunitare associated by covalent binding. In certain embodiments, β2microglobulin and α3 subunit are associated non-covalently. The α1 andα2 subunits of the heavy chain fold to form a groove for a peptide,e.g., antigenic determinant, to be displayed and recognized by TCR.

Class I molecules generally associate with, e.g., bind, peptides ofabout 8-9 amino acids (e.g., 7-11 amino acids) in length. All humanshave between three and six different class I molecules, which can eachbind many different types of peptides. In one specific embodiment, theclass I MHC polypeptide is a human class I MHC polypeptide selected fromthe group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.

In some embodiments, the targeting moiety comprises an MHC class I αheavy chain extracellular domain (human α1, α2, and/or α3 domains)without a transmembrane domain. In some embodiments, the class I α heavychain polypeptide is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, orHLA-L. In some embodiments, the HLA-A sequence can be an HLA-A*0201sequence.

The peptide in the pMHC complex can have the amino acid sequence of apeptide which can be associated with, e.g., presented by, an MHC class Imolecule. In certain embodiments, the sequence can comprise from 6 to 20contiguous amino acids. In certain embodiments, a peptide sequence canbe that of a protein fragment, wherein the protein is a derived from,e.g., a portion of, a cellular protein, such as, for example, a proteinassociated with cancer or cancer neoantigen, and wherein the peptide canbe bound to the MHC class I heavy chain.

In some embodiments, a pMHC complex targeting moiety comprises (i) anantigenic peptide; (ii) a class I MHC polypeptide or a fragment, mutantor derivative thereof (e.g., the extracellular domain), and optionally,(iii) a β2 microglobulin polypeptide or a fragment, mutant or derivativethereof. For example, the pMHC complex can comprise, from the N- toC-terminus, (i) an antigenic peptide, (ii) a β2M sequence, and (iii) aclass I α (heavy) chain sequence. Alternatively, the pMHC complex cancomprise, from the N- to C-terminus, (i) an antigenic peptide, (ii) aclass I a (heavy) chain sequence, and (iii) a β2M sequence.

In one specific embodiment, the antigenic peptide and the MHC sequenceand/or the MHC sequence and the β2M domain are linked to one another viaa peptide linker, e.g., as described in Section 6.7. In someembodiments, a single-chain pMHC complex can comprise a first flexiblelinker between the peptide segment and the β2 microglobulin segment. Forexample, linkers can extend from and connect the carboxy terminal of thepeptide to the amino terminal of the β2 microglobulin segment. In someembodiments, the linkers are structured to allow the peptide to foldinto the binding groove resulting in a functional pMHC complex. In someembodiments, this linker can comprise at least 3 amino acids, up toabout 15 amino acids (e.g., 20 amino acids). The pMHC linker cancomprise a second flexible linker inserted between the β2 microglobulinand MHC I heavy chain segment. For example, linkers can extend from andconnect the carboxy terminal of the β2 microglobulin segment to theamino terminal of the MHC I heavy chain segment. In certain embodiments,the β2 microglobulin and the MHC I heavy chain can fold into the bindinggroove resulting in a molecule which can function in promoting T cellexpansion.

When β2M is present, the pMHC complex can include mutations in β2M andin the MHC class I α heavy chain domain such that a disulfide bond mayform between them. Exemplary amino acid pairs that can be substitutedwith cysteines to allow for disulfide bonding between the two domainsare identified in Table 2 below or as described in PCT Pub. WO2015/195531, incorporated herein by reference in its entirety:

TABLE 2 β2M Domain MHC Domain 12 236 12 237 8 234 10 235 24 236 28 23298 192 99 234 3 120 31 96 53 35 60 96 60 122 63 27 R3 G120 H31 Q96 D53R35 W60 Q96 W60 D122 Y63 Y27 K6 E232 Q8 R234 Y10 P235 S11 Q242 N24 A236S28 E232 D98 H192 M99 R234 R12 G237

In further embodiments, the single-chain pMHC complex can comprise apeptide covalently attached to an MHC class I α (heavy) chain via adisulfide bridge (i.e., a disulfide bond between two cysteines). See,e.g., U.S. Pat. Nos. 8,992,937 and 8,895,020, each of which isincorporated in its entirety by reference. In certain embodiments, thedisulfide bond comprises a first cysteine, that is positioned within alinker extending from the carboxy terminal of the peptide, and a secondcysteine that is positioned within an MHC class I heavy (e.g., an MHCclass I α (heavy) chain which has a non-covalent binding site for theantigen peptide). In certain embodiments, the second cysteine can be amutation (addition or substitution) in the MHC class I a (heavy) chain.Preferably, the pMHC complex can comprise one contiguous polypeptidechain as well as a disulfide bridge. Alternatively, the pMHC complex cancomprise two contiguous polypeptide chains which are attached via thedisulfide bridge as the only covalent linkage. In some embodiments, thelinking sequences can comprise at least one amino acid in addition tothe cysteine, including one or more glycines, one or more, alanines,and/or one or more serines. In some embodiments, the single-chainmolecule comprises from N-terminus to C-terminus an MHC class I peptide(e.g., an antigenic peptide), a first linker that comprises a firstcysteine, a β2-microglobulin sequence, a second linker, and a MHC classI heavy chain sequence comprising a second cysteine, wherein the firstcysteine and the second cysteine comprise a disulfide bridge. In someembodiments, the second cysteine is a substitution of an amino acid ofthe MHC class I heavy chain selected from the group consisting of T80C,Y84C and N86C (Y84C refers to a mutation at position 108 in a matureprotein, where the mature protein lacks a signal sequence.Alternatively, when the protein still includes a 24 mer signal sequence,the position is instead referred to as Y108C).

In certain embodiments, the disulfide bridge can link a peptide in theclass I groove of the pMHC complex if the pMHC complex comprises a firstcysteine in a Gly-Ser linker extending between the C-terminus of thepeptide and the β2 microglobulin, and a second cysteine in a proximalheavy chain position.

When present, the β2 microglobulin sequence can comprise a full-length(human or non-human) β2 microglobulin sequence. In certain embodiments,the β2 microglobulin sequence lacks the leader peptide sequence. Assuch, the β2 microglobulin sequence can comprise about 99 amino acids.An exemplary human β2 microglobulin sequence is Genbank accession no.AF072097.1.

As an alternative to type I MHC-based pMHC complexes, the IL2 agonistsof the disclosure can include a class II MHC-based pMHC complexes astargeting moieties. A class II MHC-based pMHC complex generally includesa class I MHC polypeptide or a fragment, mutant or derivative thereof.In one specific embodiment, the MHC comprises α and β polypeptides of aclass II MHC molecule or a fragment, mutant or derivative thereof. Inone specific embodiment, the α and β polypeptides are linked by apeptide linker. In one specific embodiment, the MHC comprises α and βpolypeptides of a human class II MHC molecule selected from the groupconsisting of HLA-DP, HLA-DR, HLA-DQ, HLA-DM and HLA-DO.

MHC class II molecules generally consist of two polypeptide chains, αand β. The chains may come from the DP, DQ, or DR gene groups. There areabout 40 known different human MHC class II molecules. All have the samebasic structure but vary subtly in their molecular structure. MHC classII molecules bind peptides of 13-18 amino acids in length.

In some embodiments, the pMHC complex comprises one or more MHC class IIα chains or an extracellular portion thereof. In some embodiments, theclass II α chain is HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA or HLA-DRA.

In other embodiments, the pMHC complex comprises one or more MHC classII β chains or an extracellular portion thereof. In some embodiments,the class II β chain is HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB or HLA-DRB.

The peptide in a pMHC complex can be any peptide that is capable ofbinding to an MHC protein in a manner such that the pMHC complex canbind to a TCR, e.g., in a specific manner.

Examples include peptides produced by hydrolysis and most typically,synthetically produced peptides, including randomly generated peptides,specifically designed peptides, and peptides where at least some of theamino acid positions are conserved among several peptides and theremaining positions are random.

In nature, peptides that are produced by hydrolysis undergo hydrolysisprior to binding of the antigen to an MHC protein. Class I MHC typicallypresent peptides derived from proteins actively synthesized in thecytoplasm of the cell. In contrast, class II MHC typically presentpeptides derived either from exogenous proteins that enter a cell'sendocytic pathway or from proteins synthesized in the ER. Intracellulartrafficking permits a peptide to become associated with an MHC protein.

The binding of a peptide to an MHC peptide binding groove can controlthe spatial arrangement of MHC and/or peptide amino acid residuesrecognized by a TCR, or pMHC-binding protein produced by an animalgenetically modified as disclosed herein. Such spatial control is due inpart to hydrogen bonds formed between a peptide and an MHC protein.Based on the knowledge on how peptides bind to various MHCs, the majorMHC anchor amino acids and the surface exposed amino acids that arevaried among different peptides can be determined. In some embodiments,the length of an MHC-binding peptide is from 5 to 40 amino acidresidues, e.g., from 6 to 30 amino acid residues, e.g., from 8 to 20amino acid residues, e.g., between 9 and 11 amino acid residues,including any size peptide between 5 and 40 amino acids in length, inwhole integer increments (i.e., 5, 6, 7, 8, 9 . . . 40). While naturallyMHC Class II-bound peptides vary from about 9-40 amino acids, in nearlyall cases the peptide can be truncated to a 9-11 amino acid core withoutloss of MHC binding activity or T-cell recognition.

The peptides in the pMHC complexes of the disclosure typically at leasta portion, e.g., an antigenic determinant, of proteins of infectiousagents (e.g., bacterial, viral or parasitic organisms), allergens, andtumor associated proteins. Preferably, the pMHC complexes comprise anantigenic determinant of cancer cells. Exemplary antigenic determinantsof cancer cells include LCMV derived peptide gp33-41, APF (126-134),BALF (276-284), CEA (571-579), CMV pp65 (495-503), FLU-M1 (58-66), gp100(154-162), gp100 (209-217), HBV Core (18-27), Her2/neu (369-377; V2v9);HPV E7 (11-20), HPV E7 (11-19), HPV E7 (82-90), KLK4 (11-19), LMP1(125-133), MAG-A3 (112-120), NYESO1 (157-165, C165A), NYES1 (157-165,C165V), p54 WT (264-272), PAP-3 (136-143), PSMA (4-12), PSMA (135-145),Survivin (96-014), Tyrosinase (369-377, 371D), and WT1 (126-134). Anexemplary HPV E7 (11-19) peptide sequence is YMLDLQPET (SEQ ID NO:7),SEQ ID NO:537 of International Patent Publication WO 2019/005897. Anexemplary HPV E7 (82-90) peptide sequence is LLMGTLGIV (SEQ ID NO:8),SEQ ID NO:538 of International Patent Publication WO 2019/005897. Thecontents of International Patent Publication WO 2019/005897 areincorporated by reference in their entirety herein.

Other antigenic determinants of cancer cells suitable for incorporationinto the pMHC targeting moieties of the disclosure are cancerneoantigens listed in Table 3 below and their corresponding HLA allele.The neoantigens contain either mutations relative to a wild type alleleor result from the expression of a new open reading frame in cancercells, as shown in Table 1 of Fritsch et al., 2014, Cancer Immunol Res2:522-529 and incorporated by reference herein in its entirety.

TABLE 3 Gene HLA allele Neoantigen SEQ ID NO: ECGF-1 B 07:02 RPHAIRRPLAL9 ME-1 A 02:01 FLDEFMEGV 10 PLEKHM2 A 01:01 LTDDRLFTCY 11 FNDC3B A 02:01VVMSWAPPV 12 PRDX5 A 02:01 LLLDDLLVSI 13 MATN2 A 11:01 KTLTSVFQK 14DDX21 A 68:01 EAFIQPITR 15 RBAF B 07:02 RPHVPESAF 16 GAS7 A 02:01SLADEAEVYL 17 ATR A 03:01 KLYEEPLLK 18 SIRT2 A 03:01 KIFSEVTLK 19 EF2A 68:02 ETVSEQSNV 20 KIAA0223 A 02:01 VLHDDLLEA 21 GAPDH A 02:01GIVEGLITTV 22 BCL2A1 A 24:02 DYLQYVLQI 23 HSP70 A 02:01 SLFEGIDIYT 24ACTININ A 02:01 FIASNGVKLV 25 CDK12 A 11:01 CILGKLFTK 26 KIAA1440A 01:01 QTACEVLDY 27 HAUS3 A 02:01 ILNAMIAKI 28 BCL2A1 B 44:03KEFEDDIINW 29 PPP1R3B A 01:01 YTDFHCQYV 30 HB-1 B 44:03 EEKRGSLHVW 31MUM-2 B 44:02 SELFRSGLDSY 32 KIAA0205 B 44:03 AEPIDIQTW 33 GPNMB A 03:01TLDWLLQTPK 34 CSNK1A1 A 02:01 GLFGDIYLAI 35 CLPP A 02:01 ILDKVLVHL 36CTNNB1 A 24:02 SYLDSGIHF 37 SNRP116 A 03:01 KILDAVVAQK 38 OS9 B 44:03KELEGILLL 39 MYH2 A 03:01 KINKNPKYK 40 MART-2 A 01:01 FLEGNEVGKTY 41NFYC B 52:01 AQQITKTEV 42 CDK4 A 02:01 ACDPHSGHFV 43 pARF14-ORF3 A 11:01AVCPWTWLR 44 HMSD-n B 44:03 MEIFIEVFSHF 45 PANE-1 A 03:01 RVWDLPGVLK 46MUM1 B 44:02 EEKLIVVLF 47 P2x5 B 07:02 TPNQRQNVC 48

6.5. The Multimerization Moiety

6.5.1. Fc Domains

The IL2 agonists of the disclosure can include an Fc region derived fromany suitable species. In one embodiment the Fc region is derived from ahuman Fc domain. In preferred embodiments, the IL2 domain is fused to anIgG Fc molecule.

The IL2 domain may be fused to the N-terminus or the C-terminus of theIgG Fc region. As shown in the Examples, fusion to the C-terminus of theIgG Fc region maintains the IL2 domain activity to a greater extent thanwhen fused to the N-terminus of the IgG Fc.

One embodiment of the present disclosure is directed to a dimercomprising two Fc-fusion polypeptides created by fusing an IL2 domain tothe Fc region of an antibody. The dimer can be made by, for example,inserting a gene fusion encoding the fusion protein into an appropriateexpression vector, expressing the gene fusion in host cells transformedwith the recombinant expression vector, and allowing the expressedfusion protein to assemble much like antibody molecules, whereuponinterchain bonds form between the Fc moieties to yield the dimer.

The Fc domain can be derived from any suitable class of antibody,including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG(including subclasses IgG1, IgG2, IgG3 and IgG4), and IgM. In oneembodiment, the Fc domain is derived from IgG1, IgG2, IgG3 or IgG4. Inone embodiment the Fc domain is derived from IgG1. In one embodiment theFc domain is derived from IgG4.

The two Fc domains within the Fc region can be the same or differentfrom one another. In a native antibody the Fc domains are typicallyidentical, but for the purpose of producing multispecific bindingmolecules, e.g., the IL2 agonists of the disclosure, the Fc domainsmight advantageously be different to allow for heterodimerization, asdescribed in Section 6.5.1.2 below.

In native antibodies, the heavy chain Fc domain of IgA, IgD and IgG iscomposed of two heavy chain constant domains (CH2 and CH3) and that ofIgE and IgM is composed of three heavy chain constant domains (CH2, CH3and CH4). These dimerize to create an Fc region.

In IL2 agonists of the present disclosure, the Fc region, and/or the Fcdomains within it, can comprise heavy chain constant domains from one ormore different classes of antibody, for example one, two or threedifferent classes.

In one embodiment the Fc region comprises CH2 and CH3 domains derivedfrom IgG1.

In one embodiment the Fc region comprises CH2 and CH3 domains derivedfrom IgG2.

In one embodiment the Fc region comprises CH2 and CH3 domains derivedfrom IgG3.

In one embodiment the Fc region comprises CH2 and CH3 domains derivedfrom IgG4.

In one embodiment the Fc region comprises a CH4 domain from IgM. The IgMCH4 domain is typically located at the C-terminus of the CH3 domain.

In one embodiment the Fc region comprises CH2 and CH3 domains derivedfrom IgG and a CH4 domain derived from IgM.

It will be appreciated that the heavy chain constant domains for use inproducing an Fc region for the IL2 agonists of the present disclosuremay include variants of the naturally occurring constant domainsdescribed above. Such variants may comprise one or more amino acidvariations compared to wild type constant domains. In one example the Fcregion of the present disclosure comprises at least one constant domainthat varies in sequence from the wild type constant domain. It will beappreciated that the variant constant domains may be longer or shorterthan the wild type constant domain. Preferably the variant constantdomains are at least 60% identical or similar to a wild type constantdomain. In another example the variant constant domains are at least 70%identical or similar. In another example the variant constant domainsare at least 80% identical or similar. In another example the variantconstant domains are at least 90% identical or similar. In anotherexample the variant constant domains are at least 95% identical orsimilar.

IgM and IgA occur naturally in humans as covalent multimers of thecommon H2L2 antibody unit. IgM occurs as a pentamer when it hasincorporated a J-chain, or as a hexamer when it lacks a J-chain. IgAoccurs as monomer and dimer forms. The heavy chains of IgM and IgApossess an 18 amino acid extension to the C-terminal constant domain,known as a tailpiece. The tailpiece includes a cysteine residue thatforms a disulfide bond between heavy chains in the polymer, and isbelieved to have an important role in polymerization. The tailpiece alsocontains a glycosylation site. In certain embodiments, the IL2 agonistsof the present disclosure do not comprise a tailpiece.

The Fc domains that are incorporated into the IL2 agonists of thepresent disclosure may comprise one or more modifications that alter thefunctional properties of the proteins, for example, binding toFc-receptors such as FcRn or leukocyte receptors, binding to complement,modified disulfide bond architecture, or altered glycosylation patterns.Exemplary Fc modifications that alter effector function are described inSection 6.5.1.1

The Fc domains can also be altered to include modifications that improvemanufacturability of asymmetric IL2 agonists, for example by allowingheterodimerization, which is the preferential pairing of non-identicalFc domains over identical Fc domains. Heterodimerization permits theproduction of IL2 agonists in which different polypeptide components areconnected to one another by an Fc region containing Fc domains thatdiffer in sequence. Examples of heterodimerization strategies areexemplified in Section 6.5.1.2.

It will be appreciated that any of the modifications mentioned above canbe combined in any suitable manner to achieve the desired functionalproperties and/or combined with other modifications to alter theproperties of the IL2 agonists.

6.5.1.1. Fc Domains with Altered Effector Function

In some embodiments, the Fc domain comprises one or more amino acidsubstitutions that reduces binding to an Fc receptor and/or effectorfunction.

In a particular embodiment the Fc receptor is an Fcγ receptor. In oneembodiment the Fc receptor is a human Fc receptor. In one embodiment theFc receptor is an activating Fc receptor. In a specific embodiment theFc receptor is an activating human Fcγ receptor, more specifically humanFcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In oneembodiment the effector function is one or more selected from the groupof complement dependent cytotoxicity (CDC), antibody-dependentcell-mediated cytotoxicity (ADCC), antibody-dependent cellularphagocytosis (ADCP), and cytokine secretion. In a particular embodiment,the effector function is ADCC.

In one embodiment, the Fc region comprises an amino acid substitution ata position selected from the group of E233, L234, L235, N297, P331 andP329 (numberings according to Kabat EU index). In a more specificembodiment, the Fc region comprises an amino acid substitution at aposition selected from the group of L234, L235 and P329 (numberingsaccording to Kabat EU index). In some embodiments, the Fc regioncomprises the amino acid substitutions L234A and L235A (numberingsaccording to Kabat EU index). In one such embodiment, the Fc region isan Igd Fc region, particularly a human Igd Fc region. In one embodiment,the Fc region comprises an amino acid substitution at position P329. Ina more specific embodiment, the amino acid substitution is P329A orP329G, particularly P329G (numberings according to Kabat EU index). Inone embodiment, the Fc region comprises an amino acid substitution atposition P329 and a further amino acid substitution at a positionselected from E233, L234, L235, N297 and P331 (numberings according toKabat EU index). In a more specific embodiment, the further amino acidsubstitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. Inparticular embodiments, the Fc region comprises amino acid substitutionsat positions P329, L234 and L235 (numberings according to Kabat EUindex). In more particular embodiments, the Fc region comprises theamino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA” or“LALAPG”).

Typically, the same one or more amino acid substitution is present ineach of the two Fc domains of an Fc region. Thus, in a particularembodiment, each Fc domain of the Fc region comprises the amino acidsubstitutions L234A, L235A and P329G (Kabat EU index numbering), i.e. ineach of the first and the second Fc domains in the Fc region the leucineresidue at position 234 is replaced with an alanine residue (L234A), theleucine residue at position 235 is replaced with an alanine residue(L235A) and the proline residue at position 329 is replaced by a glycineresidue (P329G) (numbering according to Kabat EU index).

In one embodiment, the Fc domain is an IgG1 Fc domain, particularly ahuman IgG1 Fc domain.

Typically, the same one or more amino acid substitution is present ineach of the two Fc domains of an Fc region. Thus, in a particularembodiment, each Fc domain of the Fc region comprises the amino acidsubstitutions L234A, L235A and P329G (Kabat EU index numbering), i.e. ineach of the first and the second Fc domains in the Fc region the leucineresidue at position 234 is replaced with an alanine residue (L234A), theleucine residue at position 235 is replaced with an alanine residue(L235A) and the proline residue at position 329 is replaced by a glycineresidue (P329G) (numbering according to Kabat EU index).

In one embodiment, the Fc domain is an IgG1 Fc domain, particularly ahuman IgG1 Fc domain. In some embodiments, the IgG1 Fc domain is avariant IgG1 comprising D265A, N297A mutations (EU numbering) to reduceeffector function.

In another embodiment, the Fc domain is an IgG4 Fc domain with reducedbinding to Fc receptors. Exemplary IgG4 Fc domains with reduced bindingto Fc receptors may comprise an amino acid sequence selected from Table4 below: In some embodiments, the Fc domain includes only the boldedportion of the sequences shown below:

TABLE 4 SEQ Fc Domain Sequence ID NO: SEQ ID NO: 1 ofAsp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys 49WO2014/121087Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro ProLys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val ThrCys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln PheAsn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr LysPro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser ValLeu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr LysCys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys ThrIle Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr ThrLeu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser LeuThr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val GluTrp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr ProPro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser ArgLeu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe SerCys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln LysSer Leu Ser Leu Ser Leu Gly Lys SEQ ID NO: 2 ofAsp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 50WO2014/121087Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe LeuPhe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr ProGlu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro GluVal Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn AlaLys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg ValVal Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly LysGlu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro GlnVal Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn GlnVal Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp IleAla Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr LysThr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe LeuTyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly AsnVal Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His TyrThr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys SEQ ID NO: 30 ofAla Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 51WO2014/121087 LysSer Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp TyrPhe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu ThrSer Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly LeuTyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu GlyThr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn ThrLys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys ThrHis Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly ProSer Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu MetIle Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp ValSer Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val AspGly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu GluGln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr ValLeu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys LysVal Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr IleSer Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr ThrLeu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val SerLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile AlaVal Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn AsnTyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp GlnGln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala LeuHis Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly LysSEQ ID NO: 31 ofAla Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 52WO2014/121087Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val LysAsp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly AlaLeu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser SerGly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser SerLeu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro SerAsn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly ProPro Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro SerVal Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met IleSer Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val SerGln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp GlyVal Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu GlnPhe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val LeuHis Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys ValSer Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile SerLys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr LeuPro Pro Ser Gln Glu Glu Met Thr Lys AsnGln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro SerAsp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu AsnAsn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly SerPhe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg TrpGln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu AlaLeu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly LysSEQ ID NO: 37 ofAla Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 53WO2014/121087 LysSer Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp TyrPhe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu ThrSer Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly LeuTyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu GlyThr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn ThrLys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys ThrHis Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly ProSer Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu MetIle Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp ValSer Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val AspGly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu GluGln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr ValLeu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys LysVal Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr IleSer Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr ThrLeu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val SerLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile AlaVal Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn AsnTyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp GlnGln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala LeuHis Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly LysSEQ ID NO: 38 ofAla Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 54WO2014/121087Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val LysAsp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly AlaLeu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser SerGly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser SerLeu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro SerAsn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly ProPro Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro SerVal Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met IleSer Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val SerGln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp GlyVal Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu GlnPhe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val LeuHis Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys ValSer Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile SerLys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr LeuPro Pro Ser Gln Glu Glu Met Thr Lys AsnGln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro SerAsp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu AsnAsn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly SerPhe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg TrpGln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu AlaLeu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

In a particular embodiment, the IgG4 with reduced effector functioncomprises the bolded portion of the amino acid sequence of SEQ ID NO:31of WO2014/121087, sometimes referred to herein as IgG4s or hIgG4s.

For heterodimeric Fc regions, it is possible to incorporate acombination of the variant IgG4 Fc sequences set forth above, forexample an Fc region comprising a combination of SEQ ID NO:30 ofWO2014/121087 (or the bolded portion thereof) and SEQ ID NO:37 ofWO2014/121087 (or the bolded portion thereof) or an Fc region comprisinga combination of SEQ ID NO:31 of WO2014/121087 (or the bolded portionthereof) and SEQ ID NO:38 of WO2014/121087 (or the bolded portionthereof).

6.5.1.2. Fc Heterodimerization Variants

Certain IL2 agonists entail dimerization between two Fc domains that,unlike a native immunoglobulin, are operably linked to non-identicalN-terminal regions, e.g., one Fc domain connected to a Fab and the otherFc domain connected to an IL2 moiety. Inadequate heterodimerization oftwo Fc regions to form an Fc domain has can be an obstacle forincreasing the yield of desired heterodimeric molecules and representschallenges for purification. A variety of approaches available in theart can be used in for enhancing dimerization of Fc domains that mightbe present in the IL2 agonists of the disclosure, for example asdisclosed in EP 1870459A1; U.S. Pat. Nos. 5,582,996; 5,731,168;5,910,573; 5,932,448; 6,833,441; 7,183,076; U.S. Patent ApplicationPublication No. 2006204493A1; and PCT Publication No. WO 2009/089004A1.

The present disclosure provides IL2 agonists comprising Fc heterodimers,Fc regions comprising heterologous, non-identical Fc domains. Typically,each Fc domain in the Fc heterodimer comprises a CH3 domain of anantibody. The CH3 domains are derived from the constant region of anantibody of any isotype, class or subclass, and preferably of IgG (IgG1,IgG2, IgG3 and IgG4) class, as described in the preceding section.

Heterodimerization of the two different heavy chains at CH3 domains giverise to the desired IL2 agonist, while homodimerization of identicalheavy chains will reduce yield of the desired IL2 agonist. Thus, in apreferred embodiment, the polypeptides that associate to form an IL2agonist of the disclosure will contain CH3 domains with modificationsthat favor heterodimeric association relative to unmodified Fc domains.

In a specific embodiment said modification promoting the formation of Fcheterodimers is a so-called “knob-into-hole” or “knob-in-hole”modification, comprising a “knob” modification in one of the Fc domainsand a “hole” modification in the other Fc domain. The knob-into-holetechnology is described e.g., in U.S. Pat. Nos. 5,731,168; 7,695,936;Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, 2001, Immunol Meth248:7-15. Generally, the method involves introducing a protuberance(“knob”) at the interface of a first polypeptide and a correspondingcavity (“hole”) in the interface of a second polypeptide, such that theprotuberance can be positioned in the cavity so as to promoteheterodimer formation and hinder homodimer formation. Protuberances areconstructed by replacing small amino acid side chains from the interfaceof the first polypeptide with larger side chains (e.g., tyrosine ortryptophan). Compensatory cavities of identical or similar size to theprotuberances are created in the interface of the second polypeptide byreplacing large amino acid side chains with smaller ones (e.g., alanineor threonine).

Accordingly, in some embodiments, an amino acid residue in the CH3domain of the first subunit of the Fc domain is replaced with an aminoacid residue having a larger side chain volume, thereby generating aprotuberance within the CH3 domain of the first subunit which ispositionable in a cavity within the CH3 domain of the second subunit,and an amino acid residue in the CH3 domain of the second subunit of theFc domain is replaced with an amino acid residue having a smaller sidechain volume, thereby generating a cavity within the CH3 domain of thesecond subunit within which the protuberance within the CH3 domain ofthe first subunit is positionable. Preferably said amino acid residuehaving a larger side chain volume is selected from the group consistingof arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).Preferably said amino acid residue having a smaller side chain volume isselected from the group consisting of alanine (A), serine (S), threonine(T), and valine (V). The protuberance and cavity can be made by alteringthe nucleic acid encoding the polypeptides, e.g., by site-specificmutagenesis, or by peptide synthesis. An exemplary substitution isY470T.

In a specific such embodiment, in the first Fc domain the threonineresidue at position 366 is replaced with a tryptophan residue (T366W),and in the Fc domain the tyrosine residue at position 407 is replacedwith a valine residue (Y407V) and optionally the threonine residue atposition 366 is replaced with a serine residue (T366S) and the leucineresidue at position 368 is replaced with an alanine residue (L368A)(numbering according to Kabat EU index). In a further embodiment, in thefirst Fc domain additionally the serine residue at position 354 isreplaced with a cysteine residue (S354C) or the glutamic acid residue atposition 356 is replaced with a cysteine residue (E356C) (particularlythe serine residue at position 354 is replaced with a cysteine residue),and in the second Fc domain additionally the tyrosine residue atposition 349 is replaced by a cysteine residue (Y349C) (numberingaccording to Kabat EU index). In a particular embodiment, the first Fcdomain comprises the amino acid substitutions S354C and T366W, and thesecond Fc domain comprises the amino acid substitutions Y349C, T366S,L368A and Y407V (numbering according to Kabat EU index).

In some embodiments, electrostatic steering (e.g., as described inGunasekaran et al., 2010, J Biol Chem 285(25): 19637-46) can be used topromote the association of the first and the second Fc domain of the Fcregion.

As an alternative, or in addition, to the use of Fc domains that aremodified to promote heterodimerization, an Fc domain can be modified toallow a purification strategy that enables selections of Fcheterodimers. In one such embodiment, one polypeptide comprises amodified Fc domain that abrogates its binding to Protein A, thusenabling a purification method that yields a heterodimeric protein. See,for example, U.S. Pat. No. 8,586,713. As such, the IL2 agonists comprisea first CH3 domain and a second Ig CH3 domain, wherein the first andsecond Ig CH3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe IL2 agonist to Protein A as compared to a corresponding IL2 agonistlacking the amino acid difference. In one embodiment, the first CH3domain binds Protein A and the second CH3 domain contains amutation/modification that reduces or abolishes Protein A binding suchas an H95R modification (by IMGT exon numbering; H435R by EU numbering).The second CH3 may further comprise a Y96F modification (by IMGT; Y436Fby EU). This class of modifications is referred to herein as “star”mutations.

In some embodiments, the Fc can contain one or more mutations (e.g.,knob and hole mutations) to facilitate heterodimerization as well asstar mutations to facilitate purification.

6.6. Stabilization Moieties

The IL2 agonists of the disclosure can comprise a stabilization moietythat can extend the molecule's serum half-life in vivo. Serum half-lifeis often divided into an alpha phase and a beta phase. Either or bothphases may be improved significantly by addition of an appropriatestabilization moiety. For example, the stabilization moiety can increasethe serum half-life of the IL2 agonist by more than 5, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 120, 150, 200, 400, 600, 800, 1000% or morerelative to a corresponding IL2 agonist not containing the stabilizationmoiety. For the purpose of this disclosure, serum half-life can refer tothe half-life in humans or other mammals (e.g., mice or non-humanprimates).

Wild type IL2 has a serum half-life of less than 10 minutes. The IL2agonists of the disclosure have preferably a serum half-life in humansand/or mice of at least about 2 hours, at least about 4 hours, at leastabout 6 hours, or at least about 8 hours. In some embodiments, the IL2agonists of the disclosure have a serum half-life of at least 10 hours,at least 12 hours, at least 15 hours, at least 18 hours, at least 24hours, at least 36 hours, at least 48 hours, at least 60 hours, or atleast 72 hours.

Stabilization moieties, include polyoxyalkylene moieties (e.g.,polyethylene glycol), sugars (e.g., sialic acid), and well-toleratedprotein moieties (e.g., Fc and fragments and variants thereof,transferrin, or serum albumin).

Other stabilization moieties that can be used in the IL2 agonists of thedisclosure include those described in Kontermann et al., 2011, CurrentOpinion in Biotechnology 22:868-76. Such Stabilization moieties include,but are not limited to, human serum albumin fusions, human serum albuminconjugates, human serum albumin binders (e.g., Adnectin PKE, AlbudAb,ABD), XTEN fusions, PAS fusions (i.e., recombinant PEG mimetics based onthe three amino acids proline, alanine, and serine), carbohydrateconjugates (e.g., hydroxyethyl starch (HES)), glycosylation, polysialicacid conjugates, and fatty acid conjugates.

Accordingly, in some embodiments the disclosure provides an IL2 agonistcomprising a stabilization moiety that is a polymeric sugar.

Serum albumin can also be engaged in half-life extension through moduleswith the capacity to non-covalently interact with albumin. Accordingly,the IL2 agonists of the disclosure can include as a stabilization moietyan albumin-binding protein. The albumin-binding protein can be eitherconjugated or genetically fused to one or more other components of theIL2 agonist of the disclosure. Proteins with albumin-binding activityare known from certain bacteria. For example, streptococcal protein Gcontains several small albumin-binding domains composed of roughly 50amino acid residues (6 kDa). Additional examples of serum albuminbinding proteins such as those described in U.S. Publication Nos.2007/0178082 and 2007/0269422. Fusion of an albumin binding domain to aprotein results in a strongly extended half-life (see Kontermann et al.,2011, Current Opinion in Biotechnology 22:868-76).

In other embodiments the stabilization moiety is human serum albumin. Inother embodiments, the stabilization moiety is transferrin.

In some embodiments, the stabilization moiety is an Fc domain, forexample any of the Fc domains described in Section 6.5.1 and subsectionsthereof, incorporated by reference herein. The Fc domains described inSection 6.5.1 are generally capable of dimerization. However, for thepurpose of stabilization the Fc domain can be a soluble monomeric Fcdomain that has a reduced ability to self-associate. See, e.g., Helm etal., 1996, J. Biol. Chem. 271: 7494-7500 and Ying et al., 2012, J BiolChem. 287(23):19399-19408. An example of a soluble monomeric Fc domaincomprises amino acid substitutions in the positions corresponding toT366 and/or Y407 in CH3, as described in U.S. Patent Publication No.2019/0367611. The monomeric Fc domains can be of any Ig subtype and caninclude additional substitutions that reduce effector function, asdescribed in Section 6.5.1 and subsections thereof.

In yet other embodiments, the stabilization moiety is a polyethyleneglycol moiety or another polymer, as described in Section 6.6.1 below.

The stabilization moiety can be connected to one or more othercomponents of the IL2 agonists of the disclosure via a linker, forexample as described in Section 6.7 below.

6.6.1. Polyethylene Glycol

In some embodiments, the IL2 agonist comprises polyethylene glycol (PEG)or another hydrophilic polymer as a stabilization moiety, for example acopolymer of ethylene glycol/propylene glycol, carboxymethylcellulose,dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), dextran or poly(n-vinylpyrrolidone)polyethylene glycol, a propropylene glycol homopolymer, aprolypropylene oxide/ethylene oxide co-polymer, a polyoxyethylatedpolyol (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Thepolymer may be of any molecular weight, and may be branched orunbranched.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Theterm “PEG” is used broadly to encompass any polyethylene glycolmolecule, without regard to size or to modification at an end of thePEG, and can be represented by the formula: X—O(CH₂CH₂O)_(n)-1CH₂CH₂OH,where n is 20 to 2300 and X is H or a terminal modification, e.g., aC₁₋₄ alkyl. PEG can contain further chemical groups which are necessaryfor binding reactions, which result from the chemical synthesis of themolecule; or which act as a spacer for optimal distance of parts of themolecule. In addition, such a PEG can consist of one or more PEGside-chains which are linked together. PEGs with more than one PEG chainare called multiarmed or branched PEGs. Branched PEGs are described in,for example, European Application No. 473084A and U.S. Pat. No.5,932,462.

One or more PEG molecules can be attached at different positions on theIL2 agonist, and such attachment may be achieved by reaction withamines, thiols or other suitable reactive groups. The amine moiety maybe, for example, a primary amine found at the N-terminus of the IL2agonist (or a component thereof) or an amine group present in an aminoacid, such as lysine or arginine. In some embodiments, the PEG moiety isattached at a position on the IL2 agonist at a) the N-terminus; b)between the N-terminus and the most N-terminal alpha helix; c) a looppositioned on the face of IL2 that binds to IL2-Rβ; d) between theC-terminus and the most C-terminal alpha helix; e) a loop connecting twoalpha helices; and/or f) at the C-terminus.

PEGylation can be achieved by site-directed PEGylation, wherein asuitable reactive group is introduced into the protein to create a sitewhere PEGylation preferentially occurs. In some embodiments, the IL2agonist is modified to introduce a cysteine residue at a desiredposition, permitting site-directed PEGylation on the cysteine. Mutationscan be introduced into the coding sequence of an IL2 agonist of thedisclosure to generate cysteine residues. This might be achieved, forexample, by mutating one or more amino acid residues to cysteine.Preferred amino acids for mutating to a cysteine residue include serine,threonine, alanine and other hydrophilic residues. Preferably, theresidue to be mutated to cysteine is a surface-exposed residue.Algorithms are well-known in the art for predicting surfaceaccessibility of residues based on primary sequence or three dimensionalstructure. The three dimensional structure of IL2 is described in, e.g.,Wang et al., 2005, Science 310(5751):1159-63, and can be used toidentify surface-exposed residues that can be mutated to cysteine. Themutations can be chosen to avoid disrupting the interaction between IL2and one or more of its receptors, although in some embodiments (e.g.,where biased binding to a receptor is desired) the substitution of anamino acid with cysteine and subsequent pegylation is designed to reducebinding to one or more of the receptors, e.g., IL2-Rα or IL2-Rβ.PEGylation of cysteine residues may be carried out using, for example,PEG-maleimide, PEG-vinylsulfone, PEG-iodoacetamide, or PEG-orthopyridyldisulfide.

The PEG is typically activated with a suitable activating groupappropriate for coupling to a desired site on the polypeptide.PEGylation methods are well-known in the art and further described inZalipsky et al., “Use of Functionalized Poly(Ethylene Glycols) forModification of Polypeptides” in Polyethylene Glycol Chemistry:Biotechnical and Biomedical Applications, J. M. Harris, Plenus Press,New York (1992), and in Zalipsky, 1995, Advanced Drug Reviews 16:157-182.

PEG moieties may vary widely in molecular weight and may be branched orlinear. Typically, the weight-average molecular weight of PEG is fromabout 100 Daltons to about 150,000 Daltons. Exemplary weight-averagemolecular weights for PEG include about 20,000 Daltons, about 40,000Daltons, about 60,000 Daltons and about 80,000 Daltons. In certainembodiments, the molecular weight of PEG is 40,000 Daltons. Branchedversions of PEG having a total molecular weight of any of the foregoingcan also be used. In some embodiments, the PEG has two branches. Inother embodiments, the PEG has four branches. In another embodiment, thePEG is a bis-PEG (NOF Corporation, DE-200MA), in which twoIL2-containing polypeptide chains are conjugated.

Conventional separation and purification techniques known in the art canbe used to purify PEGylated IL2 agonists, such as size exclusion (e.g.,gel filtration) and ion exchange chromatography. Products can also beseparated using SDS-PAGE. Products that can be separated include mono-,di-, tri-, poly- and un-PEGylated IL2 agonists, as well as free PEG. Thepercentage of mono-PEG conjugates can be controlled by pooling broaderfractions around the elution peak to increase the percentage of mono-PEGin the composition. About 90% mono-PEG conjugates represent a goodbalance of yield and activity.

In some embodiments, the PEGylated IL2 agonists will preferably retainat least about 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% of thebiological activity associated with the unmodified IL2 agonist. In someembodiments, biological activity refers to its ability to bind to thehigh or intermediate affinity IL2 receptor, as assessed by K_(D),k_(on), or k_(off).

6.7. Linkers

In certain aspects, the present disclosure provides IL2 agonists inwhich two or more components of an IL2 agonist are connected to oneanother by a peptide linker. By way of example and not limitation,linkers can be used to connect (a) an IL2 moiety and a multimerizationmoiety; (b) an IL2 moiety and a targeting moiety; (c) a targeting moietyand a multimerization moiety (e.g., a Fab domain and an Fc domain); (d)different domains within an IL2 moiety (e.g., an IL2 domain and an IL-Radomain); or (e) different domains within a targeting moiety (e.g.,different components of a peptide-MHC complex or the VH and VL domainsin a scFv).

A peptide linker can range from 2 amino acids to 60 or more amino acids,and in certain aspects a peptide linker ranges from 3 amino acids to 50amino acids, from 4 to 30 amino acids, from 5 to 25 amino acids, from 10to 25 amino acids, 10 amino acids to 60 amino acids, from 12 amino acidsto 20 amino acids, from 20 amino acids to 50 amino acids, or from 25amino acids to 35 amino acids in length.

In particular aspects, a peptide linker is at least 5 amino acids, atleast 6 amino acids or at least 7 amino acids in length and optionallyis up to 30 amino acids, up to 40 amino acids, up to 50 amino acids orup to 60 amino acids in length.

In some embodiments of the foregoing, the linker ranges from 5 aminoacids to 50 amino acids in length, e.g., ranges from 5 to 50, from 5 to45, from 5 to 40, from 5 to 35, from 5 to 30, from 5 to 25, or from 5 to20 amino acids in length. In other embodiments of the foregoing, thelinker ranges from 6 amino acids to 50 amino acids in length, e.g.,ranges from 6 to 50, from 6 to 45, from 6 to 40, from 6 to 35, from 6 to30, from 6 to 25, or from 6 to 20 amino acids in length. In yet otherembodiments of the foregoing, the linker ranges from 7 amino acids to 50amino acids in length, e.g., ranges from 7 to 50, from 7 to 45, from 7to 40, from 7 to 35, from 7 to 30, from 7 to 25, or from 7 to 20 aminoacids in length.

Charged (e.g., charged hydrophilic linkers) and/or flexible linkers areparticularly preferred.

Examples of flexible linkers that can be used in the IL2 agonists of thedisclosure include those disclosed by Chen et al., 2013, Adv Drug DelivRev. 65(10): 1357-1369 and Klein et al., 2014, Protein Engineering,Design & Selection 27(10): 325-330. Particularly useful flexible linkersare or comprise repeats of glycines and serines, e.g., a monomer ormultimer of G_(n)S (SEQ ID NO:55) or SG, (SEQ ID NO:56), where n is aninteger from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In oneembodiment, the linker is or comprises a monomer or multimer of repeatof G₄S e.g., (GGGGS)_(n) (SEQ ID NO:57).

Polyglycine linkers can suitably be used in the IL2 agonists of thedisclosure. In some embodiments, a peptide linker comprises twoconsecutive glycines (2Gly), three consecutive glycines (3Gly), fourconsecutive glycines (4Gly) (SEQ ID NO:58), five consecutive glycines(5Gly) (SEQ ID NO:59), six consecutive glycines (6Gly) (SEQ ID NO:60),seven consecutive glycines (7Gly) (SEQ ID NO:61), eight consecutiveglycines (8Gly) (SEQ ID NO:62) or nine consecutive glycines (9Gly) (SEQID NO:63).

6.7.1. pMHC Linkers

For pMHC complexes, suitable linkers can range from 1 amino acid (e.g.,Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3amino acids to 12 amino acids, including 4 amino acids to 10 aminoacids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids,or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7amino acids. In addition to the linkers above, pMHC linkers includeglycine polymers (G)n, glycine-serine polymers (including, for example,(GS)n, (GSGGS)n (SEQ ID NO:64) and (GGGS)n (SEQ ID NO:65), where n is aninteger of at least one), glycine-alanine polymers, alanine-serinepolymers, and other flexible linkers known in the art. Glycine andglycine-serine polymers can be used; both Gly and Ser are relativelyunstructured, and therefore can serve as a neutral tether betweencomponents. Glycine polymers can be used; glycine accesses significantlymore phi-psi space than even alanine, and is much less restricted thanresidues with longer side chains (see Scheraga, 1992, Rev. ComputationalChem. 1 1173-142, incorporated herein in its entirety by reference).Exemplary linkers can comprise amino acid sequences including, but notlimited to, GGSG (SEQ ID NO:66), GGSGG (SEQ ID NO:67), GSGSG (SEQ IDNO:68), GSGGG (SEQ ID NO:69), GGGSG (SEQ ID NO:70), GSSSG (SEQ IDNO:71), GCGASGGGGSGGGGS (SEQ ID NO:72), GGGGSGGGGS (SEQ ID NO:73),GGGASGGGGSGGGGS (SEQ ID NO:74), GGGGSGGGGSGGGGS (SEQ ID NO:6),GGGASGGGGS (SEQ ID NO:75), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:76), GCGGS(SEQ ID NO:77) and the like. In some embodiments, a linker polypeptideincludes a cysteine residue that can form a disulfide bond with acysteine residue present in another portion of the pMHC complex. Incertain embodiments, the linker comprises the amino acid sequence GCGGS(SEQ ID NO:77). The substitution of a glycine in the G₄S linker (SEQ IDNO:57) with cysteine can result in the formation of a disulfide bond,for example an MHC targeting moiety with a corresponding cysteinesubstitution in HLA.A2 that stabilizes the MHC peptide within the MHCcomplex.

6.7.2. Hinge Sequences

In other embodiments, the IL2 agonists of the disclosure comprise alinker that is a hinge region. In particular, where an IL2 agonistcontains an immunoglobulin-based targeting moiety, the hinge can be usedto connect the targeting moiety, e.g., a Fab domain, to amultimerization domain, e.g., an Fc domain. The hinge region can be anative or a modified hinge region. Hinge regions are typically found atthe N-termini of Fc regions. The term “hinge region”, unless the contextdictates otherwise, refers to a naturally or non-naturally occurringhinge sequence that in the context of a single or monomeric polypeptidechain is a monomeric hinge domain and in the context of a dimericpolypeptide (e.g., a homodimeric or heterodimeric IL2 agonist formed bythe association of two Fc domains) can comprise two associated hingesequences on separate polypeptide chains.

A native hinge region is the hinge region that would normally be foundbetween Fab and Fc domains in a naturally occurring antibody. A modifiedhinge region is any hinge that differs in length and/or composition fromthe native hinge region. Such hinges can include hinge regions fromother species, such as human, mouse, rat, rabbit, shark, pig, hamster,camel, llama or goat hinge regions. Other modified hinge regions maycomprise a complete hinge region derived from an antibody of a differentclass or subclass from that of the heavy chain Fc region. Alternatively,the modified hinge region may comprise part of a natural hinge or arepeating unit in which each unit in the repeat is derived from anatural hinge region. In a further alternative, the natural hinge regionmay be altered by converting one or more cysteine or other residues intoneutral residues, such as serine or alanine, or by converting suitablyplaced residues into cysteine residues. By such means the number ofcysteine residues in the hinge region may be increased or decreased.Other modified hinge regions may be entirely synthetic and may bedesigned to possess desired properties such as length, cysteinecomposition and flexibility.

A number of modified hinge regions have already been described forexample, in U.S. Pat. No. 5,677,425, WO 99/15549, WO 2005/003170, WO2005/003169, WO 2005/003170, WO 98/25971 and WO 2005/003171 and theseare incorporated herein by reference.

In one embodiment, an IL2 agonist of the disclosure comprises an Fcregion in which one or both Fc domains possesses an intact hinge regionat its N-terminus.

In various embodiments, positions 233-236 within a hinge region may beG, G, G and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied,unoccupied, and unoccupied; or all unoccupied, with positions numberedby EU numbering.

In some embodiments, the IL2 agonists of the disclosure comprise amodified hinge region that reduces binding affinity for an Fcγ receptorrelative to a wild-type hinge region of the same isotype (e.g., humanIgG1 or human IgG4).

In one embodiment, the IL2 agonists of the disclosure comprise an Fcregion in which each Fc domain possesses an intact hinge region at itsN-terminus, where each Fc domain and hinge region is derived from IgG4and each hinge region comprise the modified sequence CPPC (SEQ IDNO:78). The core hinge region of human IgG4 contains the sequence CPSC(SEQ ID NO:79) compared to IgG1 that contains the sequence CPPC (SEQ IDNO:78). The serine residue present in the IgG4 sequence leads toincreased flexibility in this region, and therefore a proportion ofmolecules form disulfide bonds within the same protein chain (anintrachain disulfide) rather than bridging to the other heavy chain inthe IgG molecule to form the interchain disulfide. (Angel et al., 1993,Mol Immunol 30(1):105-108). Changing the serine residue to a proline togive the same core sequence as IgG1 allows complete formation ofinterchain disulfides in the IgG4 hinge region, thus reducingheterogeneity in the purified product. This altered isotype is termedIgG4P.

6.7.2.1. Chimeric Hinge Sequences

The hinge region can be a chimeric hinge region.

For example, a chimeric hinge may comprise an “upper hinge” sequence,derived from a human IgG1, a human IgG2 or a human IgG4 hinge region,combined with a “lower hinge” sequence, derived from a human IgG1, ahuman IgG2 or a human IgG4 hinge region.

In particular embodiments, a chimeric hinge region comprises the aminoacid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO:80) (previously disclosedas SEQ ID NO:8 of WO2014/121087, which is incorporated by reference inits entirety herein) or ESKYGPPCPPCPAPPVA (SEQ ID NO:81) (previouslydisclosed as SEQ ID NO:9 of WO2014/121087). Such chimeric hingesequences can be suitably linked to an IgG4 CH2 region (for example byincorporation into an IgG4 Fc domain, for example a human or murine Fcdomain, which can be further modified in the CH2 and/or CH3 domain toreduce effector function, for example as described in Section 6.5.1.1).

6.7.2.2. Hinge Sequences with Reduced Effector Function

In further embodiments, the hinge region can be modified to reduceeffector function, for example as described in WO2016161010A2, which isincorporated by reference in its entirety herein. In variousembodiments, the positions 233-236 of the modified hinge region are G,G, G and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied,unoccupied, and unoccupied; or all unoccupied, with positions numberedby EU numbering (as shown in FIG. 1 of WO2016161010A2). These segmentscan be represented as GGG-, GG--, G--- or ---- with “-” representing anunoccupied position.

Position 236 is unoccupied in canonical human IgG2 but is occupied by inother canonical human IgG isotypes. Positions 233-235 are occupied byresidues other than G in all four human isotypes (as shown in FIG. 1 ofWO2016161010A2).

The hinge modification within positions 233-236 can be combined withposition 228 being occupied by P. Position 228 is naturally occupied byP in human IgG1 and IgG2 but is occupied by S in human IgG4 and R inhuman IgG3. An S228P mutation in an IgG4 antibody is advantageous instabilizing an IgG4 antibody and reducing exchange of heavy chain lightchain pairs between exogenous and endogenous antibodies. Preferablypositions 226-229 are occupied by C, P, P and C respectively.

Exemplary hinge regions have residues 226-236, sometimes referred to asmiddle (or core) and lower hinge, occupied by the modified hingesequences designated GGG-(233-236), GG--(233-236), G---(233-236) and noG(233-236). Optionally, the hinge domain amino acid sequence comprisesCPPCPAPGGG-GPSVF (SEQ ID NO:82) (previously disclosed as SEQ ID NO:1 ofWO2016161010A2), CPPCPAPGG--GPSVF (SEQ ID NO:83) (previously disclosedas SEQ ID NO:2 of WO2016161010A2), CPPCPAPG---GPSVF (SEQ ID NO:84)(previously disclosed as SEQ ID NO:3 of WO2016161010A2), orCPPCPAP----GPSVF (SEQ ID NO:85) (previously disclosed as SEQ ID NO:4 ofWO2016161010A2).

The modified hinge regions described above can be incorporated into aheavy chain constant region, which typically include CH2 and CH3domains, and which may have an additional hinge segment (e.g., an upperhinge) flanking the designated region. Such additional constant regionsegments present are typically of the same isotype, preferably a humanisotype, although can be hybrids of different isotypes. The isotype ofsuch additional human constant regions segments is preferably human IgG4but can also be human IgG1, IgG2, or IgG3 or hybrids thereof in whichdomains are of different isotypes. Exemplary sequences of human IgG1,IgG2 and IgG4 are shown in FIGS. 2-4 of WO2016161010A2.

In specific embodiments, the modified hinge sequences can be linked toan IgG4 CH2 region (for example by incorporation into an IgG4 Fc domain,for example a human or murine Fc domain, which can be further modifiedin the CH2 and/or CH3 domain to reduce effector function, for example asdescribed in Section 6.5.1.1).

6.8. Nucleic Acids and Host Cells

In another aspect, the disclosure provides nucleic acids encoding theIL2 agonists of the disclosure. In some embodiments, the IL2 agonistsare encoded by a single nucleic acid. In other embodiments, for examplein the case of a heterodimeric molecule or a molecule comprising atargeting moiety composed of more than one polypeptide chain, the IL2agonists can be encoded by a plurality (e.g., two, three, four or more)nucleic acids.

A single nucleic acid can encode an IL2 agonist that comprises a singlepolypeptide chain, an IL2 agonist that comprises two or more polypeptidechains, or a portion of an IL2 agonist that comprises more than twopolypeptide chains (for example, a single nucleic acid can encode twopolypeptide chains of an IL2 agonist comprising three, four or morepolypeptide chains, or three polypeptide chains of an IL2 agonistcomprising four or more polypeptide chains). For separate control ofexpression, the open reading frames encoding two or more polypeptidechains can be under the control of separate transcriptional regulatoryelements (e.g., promoters and/or enhancers). The open reading framesencoding two or more polypeptides can also be controlled by the sametranscriptional regulatory elements, and separated by internal ribosomeentry site (IRES) sequences allowing for translation into separatepolypeptides.

In some embodiments, an IL2 agonist comprising two or more polypeptidechains is encoded by two or more nucleic acids. The number of nucleicacids encoding an IL2 agonist can be equal to or less than the number ofpolypeptide chains in the IL2 agonist (for example, when more than onepolypeptide chains are encoded by a single nucleic acid).

The nucleic acids of the disclosure can be DNA or RNA (e.g., mRNA).

In another aspect, the disclosure provides host cells and vectorscontaining the nucleic acids of the disclosure. The nucleic acids may bepresent in a single vector or separate vectors present in the same hostcell or separate host cell, as described in more detail herein below.

6.8.1. Vectors

The disclosure provides vectors comprising nucleotide sequences encodingan IL2 agonist or an IL2 agonist component described herein, for exampleone or two of the polypeptide chains of a half antibody. The vectorsinclude, but are not limited to, a virus, plasmid, cosmid, lambda phageor a yeast artificial chromosome (YAC).

Numerous vector systems can be employed. For example, one class ofvectors utilizes DNA elements which are derived from animal viruses suchas, for example, bovine papilloma virus, polyoma virus, adenovirus,vaccinia virus, baculovirus, retroviruses (Rous Sarcoma Virus, MMTV orMOMLV) or SV40 virus. Another class of vectors utilizes RNA elementsderived from RNA viruses such as Semliki Forest virus, Eastern EquineEncephalitis virus and Flaviviruses.

Additionally, cells which have stably integrated the DNA into theirchromosomes can be selected by introducing one or more markers whichallow for the selection of transfected host cells. The marker mayprovide, for example, prototropy to an auxotrophic host, biocideresistance (e.g., antibiotics), or resistance to heavy metals such ascopper, or the like. The selectable marker gene can be either directlylinked to the DNA sequences to be expressed, or introduced into the samecell by co-transformation. Additional elements may also be needed foroptimal synthesis of mRNA. These elements may include splice signals, aswell as transcriptional promoters, enhancers, and termination signals.

Once the expression vector or DNA sequence containing the constructs hasbeen prepared for expression, the expression vectors can be transfectedor introduced into an appropriate host cell. Various techniques may beemployed to achieve this, such as, for example, protoplast fusion,calcium phosphate precipitation, electroporation, retroviraltransduction, viral transfection, gene gun, lipid based transfection orother conventional techniques. Methods and conditions for culturing theresulting transfected cells and for recovering the expressedpolypeptides are known to those skilled in the art, and may be varied oroptimized depending upon the specific expression vector and mammalianhost cell employed, based upon the present description.

6.8.2. Cells

The disclosure also provides host cells comprising a nucleic acid of thedisclosure.

In one embodiment, the host cells are genetically engineered to compriseone or more nucleic acids described herein.

In one embodiment, the host cells are genetically engineered by using anexpression cassette. The phrase “expression cassette,” refers tonucleotide sequences, which are capable of affecting expression of agene in hosts compatible with such sequences. Such cassettes may includea promoter, an open reading frame with or without introns, and atermination signal. Additional factors necessary or helpful in effectingexpression may also be used, such as, for example, an induciblepromoter.

The disclosure also provides host cells comprising the vectors describedherein.

The cell can be, but is not limited to, a eukaryotic cell, a bacterialcell, an insect cell, or a human cell. Suitable eukaryotic cellsinclude, but are not limited to, Vero cells, HeLa cells, COS cells, CHOcells, HEK293 cells, BHK cells and MDCKII cells. Suitable insect cellsinclude, but are not limited to, Sf9 cells.

6.9. Pharmaceutical Compositions

6.9.1. Pharmaceutical Compositions Comprising IL2 Agonist Polypeptide

The IL2 agonists of the disclosure may be in the form of compositionscomprising the IL2 agonist and one or more carriers, excipients and/ordiluents. The compositions may be formulated for specific uses, such asfor veterinary uses or pharmaceutical uses in humans. The form of thecomposition (e.g., dry powder, liquid formulation, etc.) and theexcipients, diluents and/or carriers used will depend upon the intendeduses of the IL2 agonist and, for therapeutic uses, the mode ofadministration.

For therapeutic uses, the compositions may be supplied as part of asterile, pharmaceutical composition that includes a pharmaceuticallyacceptable carrier. This composition can be in any suitable form(depending upon the desired method of administering it to a patient).The pharmaceutical composition can be administered to a patient by avariety of routes such as orally, transdermally, subcutaneously,intranasally, intravenously, intramuscularly, intratumorally,intrathecally, topically or locally. The most suitable route foradministration in any given case will depend on the particular antibody,the subject, and the nature and severity of the disease and the physicalcondition of the subject. Typically, the pharmaceutical composition willbe administered intravenously or subcutaneously.

Pharmaceutical compositions can be conveniently presented in unit dosageforms containing a predetermined amount of an IL2 agonist of thedisclosure per dose. The quantity of IL2 agonist included in a unit dosewill depend on the disease being treated, as well as other factors asare well known in the art. Such unit dosages may be in the form of alyophilized dry powder containing an amount of IL2 agonist suitable fora single administration, or in the form of a liquid. Dry powder unitdosage forms may be packaged in a kit with a syringe, a suitablequantity of diluent and/or other components useful for administration.Unit dosages in liquid form may be conveniently supplied in the form ofa syringe pre-filled with a quantity of IL2 agonist suitable for asingle administration.

The pharmaceutical compositions may also be supplied in bulk fromcontaining quantities of IL2 agonist suitable for multipleadministrations.

Pharmaceutical compositions may be prepared for storage as lyophilizedformulations or aqueous solutions by mixing an IL2 agonist having thedesired degree of purity with optional pharmaceutically-acceptablecarriers, excipients or stabilizers typically employed in the art (allof which are referred to herein as “carriers”), i.e., buffering agents,stabilizing agents, preservatives, isotonifiers, non-ionic detergents,antioxidants, and other miscellaneous additives. See, Remington'sPharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additivesshould be nontoxic to the recipients at the dosages and concentrationsemployed.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They may be present at a wide variety ofconcentrations, but will typically be present in concentrations rangingfrom about 2 mM to about 50 mM. Suitable buffering agents for use withthe present disclosure include both organic and inorganic acids andsalts thereof such as citrate buffers (e.g., monosodium citrate-disodiumcitrate mixture, citric acid-trisodium citrate mixture, citricacid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additionally, phosphate buffers, histidinebuffers and trimethylamine salts such as Tris can be used.

Preservatives may be added to retard microbial growth, and can be addedin amounts ranging from about 0.2%-1% (w/v). Suitable preservatives foruse with the present disclosure include phenol, benzyl alcohol,meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzylammonium chloride, benzalconium halides (e.g., chloride, bromide, andiodide), hexamethonium chloride, and alkyl parabens such as methyl orpropyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.Isotonicifiers sometimes known as “stabilizers” can be added to ensureisotonicity of liquid compositions of the present disclosure and includepolyhydric sugar alcohols, for example trihydric or higher sugaralcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol andmannitol. Stabilizers refer to a broad category of excipients which canrange in function from a bulking agent to an additive which solubilizesthe therapeutic agent or helps to prevent denaturation or adherence tothe container wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglyceroland sodium thio sulfate; low molecular weight polypeptides (e.g.,peptides of 10 residues or fewer); proteins such as human serum albumin,bovine serum albumin, gelatin or immunoglobulins; hydrophylic polymers,such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose,fructose, glucose; disaccharides such as lactose, maltose, sucrose andtrehalose; and trisaccacharides such as raffinose; and polysaccharidessuch as dextran. Stabilizers may be present in amounts ranging from 0.5to 10 wt % per wt of IL2 agonist.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe added to help solubilize the glycoprotein as well as to protect theglycoprotein against agitation-induced aggregation, which also permitsthe formulation to be exposed to shear surface stressed without causingdenaturation of the protein. Suitable non-ionic surfactants includepolysorbates (20, 80, etc.), polyoxamers (184, 188 etc.), and pluronicpolyols. Non-ionic surfactants may be present in a range of about 0.05mg/mL to about 1.0 mg/mL, for example about 0.07 mg/mL to about 0.2mg/mL.

Additional miscellaneous excipients include bulking agents (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E), and cosolvents.

6.9.2. Pharmaceutical Compositions for Delivery of IL2 Agonist EncodingNucleic Acids

An IL2 agonist of the disclosure can be delivered by any method usefulfor gene therapy, for example as mRNA or through viral vectors encodingthe IL2 agonist under the control of a suitable promoter.

Exemplary gene therapy vectors include adenovirus- or AAV-basedtherapeutics. Non-limiting examples of adenovirus-based or AAV-basedtherapeutics for use in the methods, uses or compositions hereininclude, but are not limited to: rAd-p53, which is a recombinantadenoviral vector encoding the wild-type human tumor suppressor proteinp53, for example, for the use in treating a cancer (also known asGendicine®, Genkaxin®, Qi et al., 2006, Modern Oncology, 14:1295-1297);Ad5_d11520, which is an adenovirus lacking the E1B gene for inactivatinghost p53 (also called H101 or ONYX-015; see, e.g., Russell et al., 2012,Nature Biotechnology 30:658-670); AD5-D24-GM-CSF, an adenoviruscontaining the cytokine GM-CSF, for example, for the use in treating acancer (Cerullo et al., 2010, Cancer Res. 70:4297); rAd-HSVtk, areplication deficient adenovirus with HSV thymidine kinase gene, forexample, for the treatment of cancer (developed as Cerepro®, ArkTherapeutics, see e.g. U.S. Pat. No. 6,579,855; developed as ProstAtak™by Advantagene; International PCT Appl. No. WO2005/049094); rAd-TNFα, areplication-deficient adenoviral vector expressing human tumor necrosisfactor alpha (TNFα) under the control of the chemoradiation-inducibleEGR-1 promoter, for example, for the treatment of cancer (TNFerade™,GenVec; Rasmussen et al., 2002, Cancer Gene Ther. 9:951-7; Ad-IFNβ, anadenovirus serotype 5 vector from which the E1 and E3 genes have beendeleted expressing the human interferon-beta gene under the direction ofthe cytomegalovirus (CMV) immediate-early promoter, for example fortreating cancers (BG00001 and H5.110CMVhIFN-β, Biogen; Sterman et al.,2010, Mol. Ther. 18:852-860).

The nucleic acid molecule (e.g., mRNA) or virus can be formulated as thesole pharmaceutically active ingredient in a pharmaceutical compositionor can be combined with other active agents for the particular disordertreated. Optionally, other medicinal agents, pharmaceutical agents,carriers, adjuvants, diluents can be included in the compositionsprovided herein. For example, any one or more of a wetting agents,emulsifiers and lubricants, such as sodium lauryl sulfate and magnesiumstearate, as well as coloring agents, release agents, coating agents,sweetening, flavoring and perfuming agents, preservatives, antioxidants,chelating agents and inert gases also can be present in thecompositions. Exemplary other agents and excipients that can be includedin the compositions include, for example, water soluble antioxidants,such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite; oil-soluble antioxidants, such asascorbyl palmitate, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol; and metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid and phosphoric acid.

When used as adjunct therapy for adoptive cell transfer therapies, e.g.,CAR-expressing cell therapies as described in Section 6.11.1, the celltherapies, e.g., CAR-expressing cells, can be engineered to express theIL2 agonists of the disclosure. The IL2 agonist can be targeted to aspecific genomic locus, e.g., the endogenous IL2 locus or another locusthat is active in activated or dysfunctional lymphocytes, e.g., the PD-1locus, or inserted into a non-specific genomic locus. Targeting aspecific genomic locus can be achieved through gene editing, e.g., usingzinc finger proteins, the CRISPR/Cas9 system, and the like.

6.10. Therapeutic Indications and Methods of Treatment

IL2 agonists of the disclosure are useful in treating disease stateswhere stimulation of the immune system of the host is beneficial, inparticular conditions where an enhanced cellular immune response isdesirable. These may include disease states where the host immuneresponse is insufficient or deficient. Disease states for which the IL2agonists of the disclosure can be administered comprise, for example, atumor or infection where a cellular immune response would be a criticalmechanism for specific immunity. Specific disease states for which IL2agonists of the present disclosure can be employed include cancer, forexample renal cell carcinoma or melanoma; immune deficiency,specifically in HIV-positive patients, immunosuppressed patients,chronic infection and the like. The IL2 agonists of the disclosure maybe administered per se or in any suitable pharmaceutical composition.

In one aspect, IL2 agonists of the disclosure for use as a medicamentare provided. In further aspects, IL2 agonists of the disclosure for usein treating a disease are provided. In certain embodiments, IL2 agonistsof the disclosure for use in a method of treatment are provided. In oneembodiment, the disclosure provides an IL2 agonist as described hereinfor use in the treatment of a disease in a subject in need thereof. Incertain embodiments, the disclosure provides an IL2 agonist for use in amethod of treating a subject having a disease comprising administeringto the individual a therapeutically effective amount of the IL2 agonist.In certain embodiments the disease to be treated is a proliferativedisorder. In a preferred embodiment the disease is cancer. In certainembodiments the method further comprises administering to the individuala therapeutically effective amount of at least one additionaltherapeutic agent, e.g., an anti-cancer agent if the disease to betreated is cancer. In further embodiments, the disclosure provides anIL2 agonist for use in stimulating the immune system. In certainembodiments, the disclosure provides an IL2 agonist for use in a methodof stimulating the immune system in a subject comprising administeringto the individual an effective amount of the IL2 agonist to stimulatethe immune system. An “individual” according to any of the aboveembodiments is a mammal, preferably a human. “Stimulation of the immunesystem” according to any of the above embodiments may include any one ormore of a general increase in immune function, an increase in T cellfunction, an increase in B cell function, a restoration of lymphocytefunction, an increase in the expression of IL-2 receptors, an increasein T cell responsiveness, an increase in natural killer cell activity orlymphokine-activated killer (LAK) cell activity, and the like.

In a further aspect, the disclosure provides for the use of an IL2agonist of the disclosure in the manufacture or preparation of amedicament for the treatment of a disease in a subject in need thereof.In one embodiment, the medicament is for use in a method of treating adisease comprising administering to a subject having the disease atherapeutically effective amount of the medicament. In certainembodiments the disease to be treated is a proliferative disorder. In apreferred embodiment the disease is cancer. In one such embodiment, themethod further comprises administering to the individual atherapeutically effective amount of at least one additional therapeuticagent, e.g., an anti-cancer agent if the disease to be treated iscancer. In a further embodiment, the medicament is for stimulating theimmune system. In a further embodiment, the medicament is for use in amethod of stimulating the immune system in a subject comprisingadministering to the individual an amount effective of the medicament tostimulate the immune system. An “individual” according to any of theabove embodiments may be a mammal, preferably a human. “Stimulation ofthe immune system” according to any of the above embodiments may includeany one or more of a general increase in immune function, an increase inT cell function, an increase in B cell function, a restoration oflymphocyte function, an increase in the expression of IL-2 receptors, anincrease in T cell responsiveness, an increase in natural killer cellactivity or lymphokine-activated killer (LAK) cell activity, and thelike.

In a further aspect, the disclosure provides a method for treating adisease in a subject, comprising administering to said individual atherapeutically effective amount of an IL2 agonist of the disclosure. Inone embodiment a composition is administered to said individual,comprising the IL2 agonist of the disclosure in a pharmaceuticallyacceptable form. In certain embodiments the disease to be treated is aproliferative disorder. In a preferred embodiment the disease is cancer.In certain embodiments the method further comprises administering to theindividual a therapeutically effective amount of at least one additionaltherapeutic agent, e.g., an anti-cancer agent if the disease to betreated is cancer. In a further aspect, the disclosure provides a methodfor stimulating the immune system in a subject, comprising administeringto the individual an effective amount of an IL2 agonist to stimulate theimmune system. An “individual” according to any of the above embodimentsmay be a mammal, preferably a human. “Stimulation of the immune system”according to any of the above embodiments may include any one or more ofa general increase in immune function, an increase in T cell function,an increase in B cell function, a restoration of lymphocyte function, anincrease in the expression of IL-2 receptors, an increase in T cellresponsiveness, an increase in natural killer cell activity orlymphokine-activated killer (LAK) cell activity, and the like.

In certain embodiments the disease to be treated is a proliferativedisorder, preferably cancer. Non-limiting examples of cancers includebladder cancer, brain cancer, head and neck cancer, pancreatic cancer,lung cancer, breast cancer, ovarian cancer, uterine cancer, cervicalcancer, endometrial cancer, esophageal cancer, colon cancer, colorectalcancer, rectal cancer, gastric cancer, prostate cancer, blood cancer,skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.Other cell proliferation disorders that can be treated using an IL2agonist of the present disclosure include, but are not limited toneoplasms located in the: abdomen, bone, breast, digestive system,liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid,pituitary, testicles, ovary, thymus, thyroid), eye, head and neck,nervous system (central and peripheral), lymphatic system, pelvic, skin,soft tissue, spleen, thoracic region, and urogenital system. Alsoincluded are pre-cancerous conditions or lesions and cancer metastases.In certain embodiments the cancer is chosen from the group consisting ofrenal cell cancer, skin cancer, lung cancer, colorectal cancer, breastcancer, brain cancer, head and neck cancer. Similarly, other cellproliferation disorders can also be treated by the IL2 agonists of thepresent disclosure. Examples of such cell proliferation disordersinclude, but are not limited to: hypergammaglobulinemia,lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis,Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease,histiocytosis, and any other cell proliferation disease, besidesneoplasia, located in an organ system listed above. In anotherembodiment, the disease is related to autoimmunity, transplantationrejection, post-traumatic immune responses and infectious diseases(e.g., HIV). More specifically, the IL2 agonists may be used ineliminating cells involved in immune cell-mediated disorders, includinglymphoma; autoimmunity, transplantation rejection, graft-versus-hostdisease, ischemia and stroke. A skilled artisan readily recognizes thatin many cases the IL2 agonists may not provide a cure but may onlyprovide partial benefit. In some embodiments, a physiological changehaving some benefit is also considered therapeutically beneficial. Thus,in some embodiments, an amount of IL2 agonist that provides aphysiological change is considered an “effective amount” or a“therapeutically effective amount”. The subject, patient, or individualin need of treatment is typically a mammal, more specifically a human.

For the prevention or treatment of disease, the appropriate dosage of anIL2 agonist of the disclosure (when used alone or in combination withone or more other additional therapeutic agents) will depend on the typeof disease to be treated, the route of administration, the body weightof the patient, the particular IL2 agonist, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous or concurrent therapeutic interventions,the patient's clinical history and response to the IL2 agonist, and thediscretion of the attending physician. The practitioner responsible foradministration will, in any event, determine the concentration of activeingredient(s) in a composition and appropriate dose(s) for theindividual subject. Various dosing schedules including but not limitedto single or multiple administrations over various time-points, bolusadministration, and pulse infusion are contemplated herein.

A single administration of unconjugated IL2 can range from about 50,000IU/kg to about 1,000,000 IU/kg or more, more typically about 600,000IU/kg of IL2. This may be repeated several times a day (e.g., 2-3times), for several days (e.g., about 3-5 consecutive days) and then maybe repeated one or more times following a period of rest (e.g., about7-14 days). Thus, a therapeutically effective amount may comprise only asingle administration or many administrations over a period of time(e.g., about 20-30 individual administrations of about 600,000 IU/kg ofIL2 each given over about a 10-20 day period).

Similarly, the IL2 agonist is suitably administered to the patient atone time or over a series of treatments. Depending on the type andseverity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg-10mg/kg) of IL2 agonist can be an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. One typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. One exemplary dosage of the IL2 agonist would be in the rangefrom about 0.005 mg/kg to about 10 mg/kg. In other non-limitingexamples, a dose may also comprise from about 1 μg/kg/body weight, about5 μg/kg/body weight, about 10 μg/kg/body weight, about 50 μg/kg/bodyweight, about 100 μg/kg/body weight, about 200 μg/kg/body weight, about350 μg/kg/body weight, about 500 μg/kg/body weight, about 1 mg/kg/bodyweight, about 5 mg/kg/body weight, about 10 mg/kg/body weight, about 50mg/kg/body weight, about 100 mg/kg/body weight, about 200 mg/kg/bodyweight, about 350 mg/kg/body weight, about 500 mg/kg/body weight, toabout 1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 μg/kg/body weight to about 500 mg/kg/bodyweight, etc., can be administered, based on the numbers described above.Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10mg/kg (or any combination thereof) may be administered to the patient.Such doses may be administered intermittently, e.g., every week or everythree weeks (e.g., such that the patient receives from about two toabout twenty, or e.g., about six doses of the IL2 agonist). An initialhigher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

The IL2 agonists of the disclosure will generally be used in an amounteffective to achieve the intended purpose. For use to treat or prevent adisease condition, the IL2 agonists of the disclosure, or pharmaceuticalcompositions thereof, are administered or applied in a therapeuticallyeffective amount. Determination of a therapeutically effective amount iswell within the capabilities of those skilled in the art, especially inlight of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays, such as cell culture assays. Adose can then be formulated in animal models to achieve a circulatingconcentration range that includes the EC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the IL2 agonists which are sufficient to maintaintherapeutic effect. Usual patient dosages for administration byinjection range from about 0.1 to 50 mg/kg/day, typically from about 0.5to 1 mg/kg/day. Therapeutically effective plasma levels may be achievedby administering multiple doses each day. Levels in plasma may bemeasured, for example, by ELISA HPLC.

In cases of local administration or selective uptake, the effectivelocal concentration of the IL2 agonists may not be related to plasmaconcentration. One having skill in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

A therapeutically effective dose of the IL2 agonists described hereinwill generally provide therapeutic benefit without causing substantialtoxicity. Toxicity and therapeutic efficacy of an IL2 agonist can bedetermined by standard pharmaceutical procedures in cell culture orexperimental animals (see, e.g., Examples 7 and 8). Cell culture assaysand animal studies can be used to determine the LD₅₀ (the dose lethal to50% of a population) and the ED₅₀ (the dose therapeutically effective in50% of a population). The dose ratio between toxic and therapeuticeffects is the therapeutic index, which can be expressed as the ratioLD₅₀/ED₅₀. IL2 agonists that exhibit large therapeutic indices arepreferred. In one embodiment, the IL2 agonist according to the presentdisclosure exhibits a high therapeutic index. The data obtained fromcell culture assays and animal studies can be used in formulating arange of dosages suitable for use in humans. The dosage lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage may vary within this range dependingupon a variety of factors, e.g., the dosage form employed, the route ofadministration utilized, the condition of the subject, and the like. Theexact formulation, route of administration and dosage can be chosen bythe individual physician in view of the patient's condition. (See, e.g.,Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch.1, p. 1, incorporated herein by reference in its entirety).

The attending physician for patients treated with IL2 agonists of thedisclosure would know how and when to terminate, interrupt, or adjustadministration due to toxicity, organ dysfunction, and the like.Conversely, the attending physician would also know to adjust treatmentto higher levels if the clinical response were not adequate (precludingtoxicity). The magnitude of an administered dose in the management ofthe disorder of interest will vary with the severity of the condition tobe treated, with the route of administration, and the like. The severityof the condition may, for example, be evaluated, in part, by standardprognostic evaluation methods. Further, the dose and perhaps dosefrequency will also vary according to the age, body weight, and responseof the individual patient.

Due to lower toxicity, the IL2 agonists of the disclosure can be havehigher maximum therapeutic doses than wild type IL2, although, IL2agonists containing a stabilization moiety are typically administered atlower doses than wild type IL2 due to the prolonged half-lives.

6.11. Combination Therapy

The IL2 agonists according to the disclosure may be administered incombination with one or more other agents in therapy. For instance, anIL2 agonist of the disclosure may be co-administered with at least oneadditional therapeutic agent. The term “therapeutic agent” encompassesany agent administered to treat a symptom or disease in a subject inneed of such treatment. Such additional therapeutic agent may compriseany active ingredients suitable for the particular indication beingtreated, preferably those with complementary activities that do notadversely affect each other. In certain embodiments, an additionaltherapeutic agent is an immunomodulatory agent, a cytostatic agent, aninhibitor of cell adhesion, a cytotoxic agent, an activator of cellapoptosis, or an agent that increases the sensitivity of cells toapoptotic inducers. In a particular embodiment, the additionaltherapeutic agent is an anti-cancer agent, for example a microtubuledisruptor, an antimetabolite, a topoisomerase inhibitor, a DNAintercalator, an alkylating agent, a hormonal therapy, a kinaseinhibitor, a receptor antagonist, an activator of tumor cell apoptosis,or an antiangiogenic agent.

Such other agents are suitably present in combination in amounts thatare effective for the purpose intended. The effective amount of suchother agents depends on the amount of IL2 agonist used, the type ofdisorder or treatment, and other factors discussed above. The IL2agonists are generally used in the same dosages and with administrationroutes as described herein, or about from 1 to 99% of the dosagesdescribed herein, or in any dosage and by any route that isempirically/clinically determined to be appropriate.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate compositions), and separate administration, in which case,administration of the IL2 agonist of the disclosure can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant. IL2 agonists of the disclosure canalso be used in combination with radiation therapy.

6.11.1. Combination Therapy Using IL2 Agonist Therapy and Immunotherapy

The IL2 agonists of the disclosure can be advantageously used incombination with chimeric antigen receptor (“CAR”)-expressing cells,e.g., CAR-expressing T (“CAR-T”) cells, for example CAR-T in thetreatment of cancer or autoimmune diseases. In some embodiments, theCAR-T cells are recognized by a targeting moiety in the IL2 agonist. Thetargeting moiety can recognize a T cell receptor or another cell surfacemolecule on the CART cells. In some embodiments, a targeting moiety inthe IL2 agonist is capable of binding to an extracellular domain of theCAR, for example the antigen binding domain.

Conditioning or lymphodepletion therapy, e.g., a regimen ofcyclophosphamide and fludarabine, can also be administered to a subjectreceiving CAR and IL2 agonist therapy. Such therapy is usually performedin the days prior to administration of the CAR-expressing cells to thesubject. For example, cyclophosphamide can be administered for two days,e.g., at days −8 and −7 prior to infusion with CAR-expressing cells (theinfusion day being zero) and fludarabine can be administered to thesubject for five consecutive days from day −6 to day −2. In oneembodiment, 60 mg/kg of cyclophosphamide is administered to the subject.In one embodiment, 25 mg/m² of fludarabine is administered to thesubject. In one embodiment, there is a day of no treatment on day −1,the day immediately prior to the CAR-expressing cell infusion to thesubject.

The CAR-expressing cells can be administered in an amount ranging from10⁴ to 10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. T-cellcompositions may also be administered multiple times at these dosages.In some embodiments, the CAR-expressing cells are administered in dosesof 1×10⁶ to 1×10¹¹ cells or 1×10⁷ to 1×10⁸ cells.

The CAR-expressing cells can be activated with an anti-CD3 and/oranti-CD28 antibody in concert with IL2 expansion prior to administrationto the human subject.

The CAR-expressing cells, e.g., T cells, are preferably autologous tothe subject but can also be of allogeneic origin.

In one embodiment, the IL2 agonist is administered to the human subjectby bolus infusion for four consecutive days beginning on the day ofadministration of the population of CAR-expressing cells.

In one embodiment, the IL2 agonist is administered to the human subjectby bolus for at least five consecutive days beginning on the day ofadministration of the population of CAR-expressing cells.

The IL2 agonist can be administered for longer periods, for example fora week, for two weeks, for one month or longer. The dosing frequency canbe reduced, e.g., after exhaustion of the CAR-expressing cells. Forexample, the IL2 agonist can be initially administered on a daily basisand then the dosing frequency reduced to weekly.

The initiation of IL2 therapy can begin on the same day as, or one, two,three, four, five, six days or a week after, administration of theCAR-expressing cells.

In one embodiment, the population of cells comprises T-cells obtainedfrom the subject that have been engineered to recombinantly express theCAR.

In one embodiment, the IL2 agonist plasma level is maintained for one totwo weeks following administration of the population of cells to thesubject.

In one embodiment, the IL2 agonist plasma level is maintained for amonth following administration of the population of cells to thesubject.

6.11.1.1. CAR Components

A typical CAR comprises an extracellular region comprising antigenbinding domain, e.g., an antigen binding domain of an antibody, linkedto an intracellular signaling block that includes CD3 signaling domainsthat induce T cell activation following antigen binding (e.g., a CD3signaling region of a T cell receptor). The antigen binding domain canbe in the form of an scFv, as described in Section 6.4.2.1.

The antigen binding domain is typically linked to the signal domain viaa linker (e.g., a linker as described in Section 6.7), an optionalspacer (e.g., as described in Section 6.11.1.1.1), an optional hinge(e.g., as described in Section 6.11.1.1.2), a transmembrane domain(e.g., as described in Section 6.11.1.1.3), and an intracellularsignaling block (e.g., as described in Section 6.11.1.1.4).

6.11.1.1.1. Spacer Domain

In particular embodiments, the antigen binding domain of the CAR(followed by an optional linker) is followed by one or more “spacerdomains,” which refers to the region that moves the antigen bindingdomain away from the effector cell surface to enable proper cell/cellcontact, antigen binding and activation (Patel et al., 1999, GeneTherapy 6:412-419). The spacer domain may be derived either from anatural, synthetic, semi-synthetic, or recombinant source. In certainembodiments, a spacer domain is a portion of an immunoglobulin,including, but not limited to, one or more heavy chain constant regions,e.g., CH2 and CH3. The spacer domain can include the amino acid sequenceof a naturally occurring immunoglobulin hinge region or an alteredimmunoglobulin hinge region.

In one embodiment, the spacer domain comprises the CH2 and CH3 domainsof IgG1 or IgG4.

6.11.1.1.2. Hinge Domain

The antigen binding domain of the CAR is generally followed by one ormore “hinge domains” (downstream of the optional linker and/or spacer),which play a role in positioning the antigen binding domain away fromthe effector cell surface to enable proper cell/cell contact, antigenbinding and activation. A CAR generally comprises one or more hingedomains between the binding domain and the transmembrane domain (TM).The hinge domain may be derived either from a natural, synthetic,semi-synthetic, or recombinant source. The hinge domain can include theamino acid sequence of a naturally occurring immunoglobulin hinge regionor an altered immunoglobulin hinge region.

An “altered hinge region” refers to (a) a naturally occurring hingeregion with up to 30% amino acid changes (e.g., up to 25%, 20%, 15%,10%, or 5% amino acid substitutions or deletions), (b) a portion of anaturally occurring hinge region that is at least 10 amino acids (e.g.,at least 12, 13, 14 or 15 amino acids) in length with up to 30% aminoacid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acidsubstitutions or deletions), or (c) a portion of a naturally occurringhinge region that comprises the core hinge region (which may be 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15, or at least 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 amino acids in length). In certain embodiments,one or more cysteine residues in a naturally occurring immunoglobulinhinge region may be substituted by one or more other amino acid residues(e.g., one or more serine residues). An altered immunoglobulin hingeregion may alternatively or additionally have a proline residue of awild type immunoglobulin hinge region substituted by another amino acidresidue (e.g., a serine residue).

Other illustrative hinge domains suitable for use in the CARs describedherein include the hinge region derived from the extracellular regionsof type 1 membrane proteins such as CD8a, CD4, CD28 and CD7, which maybe wild-type hinge regions from these molecules or may be altered. Inanother embodiment, the hinge domain comprises a CD8a hinge region.

6.11.1.1.3. Transmembrane (TM) Domain

The “transmembrane domain” is the portion of the CAR that fuses theextracellular binding portion and intracellular signaling domain andanchors the CAR to the plasma membrane of the immune effector cell. Asused herein, the term “transmembrane domain” refers to any polypeptidestructure that is thermodynamically stable in a cell membrane,preferably a eukaryotic cell membrane (e.g., a mammalian cell membrane).

The TM domain may be derived either from a natural, synthetic,semi-synthetic, or recombinant source. The TM domain may be derived from(e.g., comprise at least the transmembrane region(s) of) the alpha, betaor zeta chain of the T-cell receptor, CD3E, CD3, CD4, CD5, CD8a, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137,CD152, CD 154, or PD1. In a particular embodiment, the TM domain issynthetic and predominantly comprises hydrophobic residues such asleucine and valine.

In certain embodiments, the CAR comprises a CD3 transmembrane domain(e.g., a transmembrane domain that comprises the amino acid sequenceLCYLLDGILFIYGVILTALFL (SEQ ID NO:86) or LDPKLCYLLDGILFIYGVILTALFLRVK(SEQ ID NO:87)), a CD28 transmembrane domain (e.g., a transmembranedomain that comprises the amino acid sequenceFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:88)) or a CD8a transmembranedomain (e.g., a transmembrane domain that comprises the amino acidsequence KPTTTPAPRPPTPAPTIASQPLSLR PEACRPAAGGAVHTRGLDFA (SEQ ID NO:89)).

The TM can be followed by a short linker, preferably 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 amino acids in length that links the TM domain and theintracellular signaling domain of the CAR. A glycine-serine based linker(e.g., a linker according to Section 6.7) provides a particularlysuitable linker.

6.11.1.1.4. Intracellular Signaling Domain

CARs typically comprise an intracellular signaling domain. An“intracellular signaling domain,” refers to the part of a CAR thatparticipates in transducing the message of effective antigen bindinginto the interior of the immune effector cell to elicit effector cellfunction, e.g., activation, cytokine production, proliferation andcytotoxic activity, including the release of cytotoxic factors to theCAR-bound target cell, or other cellular responses elicited with antigenbinding to the extracellular CAR domain.

The term “effector function” refers to a specialized function of animmune effector cell. Effector function of the T cell, for example, maybe cytolytic activity or help or activity including the secretion of acytokine. Thus, the term “intracellular signaling domain” refers to theportion of a protein which transduces the effector function signal andthat directs the cell to perform a specialized function. While usuallythe entire intracellular signaling domain can be employed, in many casesit is not necessary to use the entire domain. To the extent that atruncated portion of an intracellular signaling domain is used, suchtruncated portion may be used in place of the entire domain as long asit transduces the effector function signal. The term intracellularsignaling domain is meant to include any truncated portion of theintracellular signaling domain sufficient to transducing effectorfunction signal.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of intracellular signalingdomains: primary signaling domains that initiate antigen-dependentprimary activation through the TCR (e.g., a TCR/CD3 complex) andco-stimulatory signaling domains that act in an antigen-independentmanner to provide a secondary or co-stimulatory signal. In preferredembodiments, a CAR contemplated herein comprises an intracellularsignaling domain that comprises one or more “co-stimulatory signalingdomain” and a “primary signaling domain.”

Primary signaling domains regulate primary activation of the TCR complexeither in a stimulatory way, or in an inhibitory way. Primary signalingdomains that act in a stimulatory manner may contain signaling motifswhich are known as immunoreceptor tyrosine-based activation motifs orITAMs.

Illustrative examples of ITAM containing primary signaling domains thatare of particular use in the methods of the disclosure include thosederived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a,CD79b, and CD66d. In particular preferred embodiments, a CAR comprises aCD3ζ primary signaling domain and one or more co-stimulatory signalingdomains. The intracellular primary signaling and co-stimulatorysignaling domains may be linked in any order in tandem to the carboxylterminus of the transmembrane domain.

CARs contemplated herein comprise one or more co-stimulatory signalingdomains to enhance the efficacy and expansion of T cells expressing CARreceptors. As used herein, the term, “co-stimulatory signaling domain,”or “co-stimulatory domain,” refers to an intracellular signaling domainof a co-stimulatory molecule. Co-stimulatory molecules are cell surfacemolecules other than antigen receptors or Fc receptors that provide asecond signal required for efficient activation and function of Tlymphocytes upon binding to antigen. Illustrative examples of suchco-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30,CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1),CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1),CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70.

In some embodiments, the CD3 signaling region is linked toco-stimulatory endodomains of CD28, 4-1BB (also known as CD137), CD70,or OX40 (also known as CD134), or combinations thereof, or have twosignaling domains of CD3ζ in tandem. These endodomains allow for robustT cell activation during TCR recognition by antigen-presenting cells(APCs), improving cytokine production and proliferation of CAR-T cells.

In another embodiment, a CAR comprises CD28 and CD137 co-stimulatorysignaling domains and a CD3ζ primary signaling domain.

In yet another embodiment, a CAR comprises CD28 and CD134 co-stimulatorysignaling domains and a CD3ζ primary signaling domain.

In one embodiment, a CAR comprises CD137 and CD134 co-stimulatorysignaling domains and a CD3ζ primary signaling domain.

Exemplary CD3ζ signaling regions can comprise one of the following aminoacid sequences:

(SEQ ID NO: 90) LDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. (SEQ ID NO: 91)RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR

Exemplary CD28 signaling regions can comprise one of the following aminoacid sequences:

(SEQ ID NO: 92) KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRS.(SEQ ID NO: 93) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAP PRDFAAYRS(SEQ ID NO: 94) KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 88)FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 93)RSKRSRLLHSDY MNMTPRRPGPTRKHYQPYAPPRDFAAYRS

Exemplary CD137 (41BB) signaling regions can comprise the followingamino acid sequence:

(SEQ ID NO: 95) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

6.11.1.1.5. Tag

In some embodiments, the CAR comprises a tag used for identification ofthe CAR, for example a V5 epitope tag is derived from a small epitope(Pk) present on the P and V proteins of the paramyxovirus of simianvirus 5 (SV5). The V5 tag is usually used with all 14 amino acids(GKPIPNPLLGLDST) (SEQ ID NO:96), although it has also been used with ashorter 9 amino acid sequence (IPNPLLGLD) (SEQ ID NO:97).

6.11.1.1.6. Signal Peptide

In some embodiments, the CAR comprises a signal peptide. Signal peptidesfacilitate the expression of the CAR of the cell surface. Signalpeptides, including signal peptides of naturally occurring proteins orsynthetic, non-naturally occurring signal peptides, that are compatiblefor use in the CARs described herein will be evident to those of skillin the art. In some embodiments, the signal peptide is disposedN-terminus of the antigen-binding portion of the CAR. Upon expressionand processing of the CAR in a cell, e.g., a T cell, the signal peptideis cleaved and is therefore typically not present in the maturemolecule.

6.11.1.2. Preparation of CART Cells

For generating CART cells ex vivo, PBMC, peripheral blood lymphocytes,or T cells enriched therefrom, can be expanded prior to and/or followingintroduction of a nucleic encoding the CAR into the cells, for exampleby viral transduction.

T cells useful for generating CART cells can be isolated from peripheralblood lymphocytes by lysing the red blood cells and depleting themonocytes, for example, by centrifugation through a PERCOLL™ gradient orby counterflow centrifugal elutriation. A specific subpopulation of Tcells, such as CD3+, CD28⁺, CD4+, CD8+, CD45RA+, and CD45RO+ T cells,can be further isolated by positive or negative selection techniques.For example, in one embodiment, T cells are isolated by incubation withanti-CD3/anti-CD28 (e.g., 3×28)-conjugated beads, such as DYNABEADS®M-450 CD3/CD28 T, for a time period sufficient for positive selection ofthe desired T cells. In one embodiment, the time period is about 30minutes. In a further embodiment, the time period ranges from 30 minutesto 36 hours or longer and all integer values there between. In a furtherembodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. Inyet another preferred embodiment, the time period is 10 to 24 hours. Inone preferred embodiment, the incubation time period is 24 hours. Forisolation of T cells from patients with leukemia, use of longerincubation times, such as 24 hours, can increase cell yield. Longerincubation times may be used to isolate T cells in any situation wherethere are few T cells as compared to other cell types, such in isolatingtumor infiltrating lymphocytes (TIL) from tumor tissue or fromimmune-compromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells, subpopulations of T cells can be preferentially selected for oragainst at culture initiation or at other time points during theprocess. Additionally, by increasing or decreasing the ratio of anti-CD3and/or anti-CD28 antibodies on the beads or other surface,subpopulations of T cells can be preferentially selected for or againstat culture initiation or at other desired time points. The skilledartisan would recognize that multiple rounds of selection can also beused in the context of this disclosure. In certain embodiments, it maybe desirable to perform the selection procedure and use the “unselected”cells in the activation and expansion process. “Unselected” cells canalso be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4+ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD11b, CD16,HLA-DR, and CD8. T reg cells can be also be depleted by anti-C25conjugated beads or other similar method of selection.

In certain embodiments, for example for applications involving thetreatment of autoimmune disease as described in Section 6.11.1.4, it maybe desirable to enrich for or positively select for regulatory T cellswhich typically express CD4+, CD25+, CD62L^(hi), GITR⁺, and FoxP3⁺. Tregs can also be induced by recombinant expression of FoxP3.

Whether prior to or after genetic modification of the T cells to expressa desirable CAR, the T cells can be activated and expanded generallyusing methods known in the art, for example as described in U.S. Pat.Nos. 7,144,575; 7,067,318; 7,172,869; 7,232,566; or 7,175,843.

Generally, the T cells useful in the methods of the disclosure areexpanded by contact with a surface having attached thereto an agent thatstimulates a CD3/TCR complex associated signal and a ligand thatstimulates a co-stimulatory molecule on the surface of the T cells. Inparticular, T cell populations may be stimulated by contact with ananti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2antibody immobilized on a surface, or by contact with a protein kinase Cactivator (e.g., bryostatin) in conjunction with a calcium ionophore.For costimulation of an accessory molecule on the surface of the Tcells, a ligand that binds the accessory molecule is used. For example,a population of T cells can be contacted with an anti-CD3 antibody andoptionally an anti-CD28 antibody, for example on anti-CD3 and anti-CD28bead, under conditions appropriate for stimulating proliferation of theT cells.

Prior to administration to the human subject the CAR-expressing cellscan also be conditioned with IL12 (see, e.g., Emtage et al., 2003, J.Immunother. 16(2): 97-106, incorporated herein by reference).

6.11.1.3. Cancer Immunotherapy

Accordingly, the present disclosure provides methods of treating cancerin a human subject in need thereof, comprising administering to thesubject an effective amount of an IL2 agonist of the disclosure andadministering to the CAR-expressing cells, e.g., the CAR-expressing Tcells (or “CART cells”). Particularly useful T cell subtypes for thetreatment of cancer are T cells with robust CAR mediated cytotoxicity,e.g., CD3+CD8+ T cells, which can be prepared as described in 6.11.1.2above.

For treatment of cancer, the extracellular domain of the CAR can targeta tumor associated antigen, for example as described in Section 6.4.1.In certain embodiments, the tumor associated antigen is CD20, EGFR,FITC, CD19, CD22, CD33, PSMA, GD2, EGFR variants, ROR1, c-Met, HER2,CEA, mesothelin, GM2, CD7, CD10, CD30, CD34, CD38, CD41, CD44, CD74,CD123 CD133, CD171, MUC16, MUC1, CS1 (CD319), IL-13Ra2, BCMA, Lewis Y,IgG kappa chain, folate receptor-alpha, PSCA, or EpCAM. In particularembodiments:

-   -   the CAR is designed to target CD22 to treat diffuse large B-cell        lymphoma.    -   the CAR is designed to target mesothelin to treat mesothelioma,        pancreatic cancer, ovarian cancer, and the like.    -   the CAR is designed to target CD33/IL3Ra to treat acute        myelogenous leukemia and the like.    -   the CAR is designed to target c-Met to treat triple negative        breast cancer, non-small cell lung cancer, and the like.    -   the CAR is designed to target PSMA to treat prostate cancer and        the like.    -   the CAR is designed to target Glycolipid F77 to treat prostate        cancer and the like.    -   the CAR is designed to target EGFRvIII to treat glioblastoma and        the like.    -   the CAR is designed to target GD-2 to treat neuroblastoma,        melanoma, and the like.    -   the CAR is designed to target NY-ESO-1 TCR to treat myeloma,        sarcoma, melanoma, and the like.    -   the CAR is designed to target MAGE A3 TCR to treat myeloma,        sarcoma, melanoma, and the like.

Particularly useful for CAR-IL2 agonist combination therapy are IL2agonists comprising a targeting moiety that recognizes a cell surfaceantigen present on the surface of the CAR-expressing lymphocytes cells.

Where the IL2 agonist administered in combination with the CAR therapyhas a peptide-MHC targeting moiety, the CAR-expressing cell ispreferably a CD4⁺ or CD8⁺ cell whose TCR recognizes the peptide-MHCcomplex. For example, the CAR-expressing cells can be T lymphocyteswhose TCR specifically binds a pMHC complex present on the IL2 agonist,resulting in functional activation and survival of the CAR-expressingcells.

In some embodiments, the CAR itself comprises an antigen binding domain(e.g., an scFv domain) that targets a pMHC complex in the IL2 agonist,for example any pMHC fusion described in Section 6.4.3. Exemplary CARsthat target a pMHC complex with an HPV peptide (e.g., peptidescorresponding to amino acid residues 11-19 or 82-90 of the HPV16E7polypeptide) are disclosed in U.S. Pat. No. 10,806,780 B2, which isincorporated by reference herein in its entirety. In some embodiments,the CAR comprises a heavy chain variable domain (VH) and light chainvariable domain (VL) amino acid sequence pair selected from any one ofSEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122,130/138, 146/154, 162/170, 178/186, 194/202, 210/202, 218/226, 234/242,250/258, 266/274, 282/290, 298/306, 314/322, 330/338, 346/354, 362/370,378/386, 394/402, 410/418, 426/434, 442/450, 458/466, 474/482, 490/498,506/514, and 522/530, in each case of U.S. Pat. No. 10,806,780 B2, eachof which is incorporated by reference herein. In particular embodiments,the VH-VL pair comprise SEQ ID NOs. 2 and 10 of U.S. Pat. No. 10,806,780B2, SEQ ID NOs. 34 and 42 of U.S. Pat. No. 10,806,780 B2, SEQ ID NOs. 82and 90 of U.S. Pat. No. 10,806,780 B2, SEQ ID NOs. 194 and 202 of U.S.Pat. No. 10,806,780 B2, or SEQ ID NOs. 506 and 514 of U.S. Pat. No.10,806,780 B2, each of which is incorporated by reference herein.CAR-IL2 agonist combination therapy with CARs targeting a pMHC with anHPV peptide can be used to treat an HPV-positive cancer, e.g., squamouscell carcinoma, e.g., cervical cancer, head and neck small cellcarcinoma, anogenital cancer, and oropharyngeal cancer.

6.11.1.4. Immunotherapy of Autoimmune Disease

Chimeric antigen receptor (CAR) T cells have become powerful treatmentoptions for blood cancers. By using the same idea of modifying T cellsto efficiently target disease calls, scientists have efficientlyengineered T cells with a predetermined antigen-specificity viatransfection of viral vectors encoding chimeric antigen receptors(CARs). CAR-modified T cells engineered in a non-MHC restricted mannerhave the advantage of widespread applications, especially intransplantation and autoimmunity.

CAR-expressing Treg cells can be prepared as described in 6.11.1.2above.

Particularly useful for CAR-IL2 agonist combination therapy are IL2agonists comprising a targeting moiety that recognizes a cell surfaceantigen present on the surface of the CAR-expressing lymphocytes cells,e.g., a pMHC cloned from an autoimmune target cell.

For use in treatment of autoimmune disease, the extracellular domain ofthe CAR is preferably specific for a target antigen or ligand associatedwith the autoimmune response. Such modification causes activation ofredirected Tregs at sites of inflammation to suppress theproinflammatory effector-type immune responses. Examples of autoimmunediseases targeted by CAR T_(reg) therapy are multiple sclerosis,inflammatory bowel diseases (IBD), rheumatoid arthritis, systemic lupuserythematosus, Crohn's Disease, psoriasis, Type I Diabetes, Sjogren'sdisease, myasthenia gravis (MG), Hashimoto's thyroiditis; Graves'Disease, and uveitis.

In particular embodiments, the Tregs are engineered to express a CARtargeting an antigen or ligand specific to:

-   -   inflammatory bowel disease (IBD), wherein the antigen or ligand        is one that is expressed in diseased colon or ileum;    -   rheumatoid arthritis, wherein the antigen or ligand is an        epitope of collagen or an antigen present in joints;    -   Type I diabetes mellitus or autoimmune insulitis, wherein the        antigen or ligand is a pancreatic β cell antigen;    -   multiple sclerosis, wherein the antigen or ligand is, for        example, a myelin basic protein (MBP) antigen or MOG-1 or        MOG2-2, or a neuronal antigen;    -   autoimmune thyroiditis, wherein the antigen or ligand is a        thyroid antigen;    -   autoimmune gastritis, wherein the antigen or ligand is a gastric        antigen;    -   autoimmune uveitis or uveoretinitis, wherein the antigen or        ligand is S-antigen or another uveal or retinal antigen;    -   autoimmune orchitis, wherein the antigen or ligand is a        testicular antigen;    -   autoimmune oophoritis, wherein the antigen or ligand is an        ovarian antigen;    -   psoriasis, wherein the antigen or ligand is a keratinocyte        antigen or another antigen present in dermis or epidermis;    -   vitiligo, where the antigen or ligand is a melanocyte antigen        such as melanin or tyrosinase;    -   autoimmune prostatitis, wherein the antigen or ligand is a        prostate antigen;    -   any undesired immune response, wherein the antigen or ligand is        an activation antigen or other antigen expressed on T effector        cells present at the site of the undesired response;    -   tissue rejection, wherein the antigen or ligand is the MHC        specific to the transplanted tissue; and    -   an inflammatory condition, wherein the antigen or ligand is one        that is expressed on nonlymphoid cells of the hemopoietic        lineage that participate in inflammation.

In one embodiment, T cells can be engineered to express chimericautoantigen receptor (CAAR) T cells to specifically eliminate B cellsthat are responsible for autoimmune diseases. Accordingly, reference toCAR-expressing T cells includes reference to CAAR-expressing unless thecontext dictates otherwise.

7. EXAMPLES

7.1. Materials and Methods

7.1.1. Production of IL2 and IL15 Agonists

Constructs encoding the IL2 and IL15 muteins (identified with an IL2M_or IL15M_ designation, as appropriate), with or without a targetingmoiety (identified with a T designation), described (or containingmodules described) in Tables 5A and 5B below, as well as Fc controlswere generated. The constructs were expressed in Expi293F™ cells bytransient transfection (Thermo Fisher Scientific). Proteins in Expi293Fsupernatant were purified using the ProteinMaker system (ProteinBioSolutions, Gaithersburg, Md.) with HiTrap Protein G HP columns (GEHealthcare). After single step elution, the muteins were neutralized,dialyzed into a final buffer of phosphate buffered saline (PBS) with 5%glycerol, aliquoted and stored at −80° C.

TABLE 5A Molecule/ Alternate Module Name Description Sequence IL2 IL2Recombinant human IL2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYM(source: BD Pharmingen ™)PKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 2) IL2M0 IL2Human IL2 (WT)- GGGGS-Fc APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMMutein 0 Fc is from SEQ ID NO: 31 ofPKKATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVWO2014/121087, which is anLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSESIgG4 with reduced effector KYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVfunction SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK (SEQ ID NO: 98) IL2M1IL2 CD122-biased human IL2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYM Mutein 1(F42A, Y45A, L72G)- GGGGS-FcPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLFc is from SEQ ID NO: 31 ofELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSESKWO2014/121087, which is an YGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSIgG4 with reduced effector QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQfunction DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK (SEQ ID NO: 99) IL2M2IL2 Human IL2-(GGGGS)₄-human IL-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYM Mutein 2 2Ra-GGGGS-FcPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLIL2 interacts with IL-2Rα inELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSGGthis fusion protein to form GGSGGGGSGGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECinactive head-to-tail dimers KRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVT that slowly dissociate intoPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIan active monomer, with ~99.5%YHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLof a population believed to beICTGGGGSESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTin the inactive dimeric form.PEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY Fc is from SEQ ID NO: 31 ofRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE WO2014/121087, which is anPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN IgG4 with reduced effectorNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH functionNHYTQKSLSLSLGK (SEQ ID NO: 100) IL2M3 IL2 Fc-(GGGGS)3-human IL2-Rα-ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV Mutein 3(GGGGS)5-human IL2 fusion DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVIL2 interacts with IL-2Rα inLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPthis fusion protein to formSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLinactive head-to-tail dimersDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS that slowly dissociate intoLSLGKGGGGSGGGGSGGGGSELCDDDPPEIPHATFKAMAYKEGan active monomer, with ~99.5%TMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRof a population believed to beNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWin the inactive dimeric form.ENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKT Fc is from SEQ ID NO: 31 ofRWTQPQLICTGGGGGSGGGGSGGGGSGGGGSGGGGSAPTSSS WO2014/121087, which is anTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELIgG4 with reduced effectorKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSE functionTTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 101) IL2M4 IL2Fc-GGGGS-human IL2(C125A) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVMutein 4 IL2(C125A) is an IL2 variant inSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHwhich cysteine 125 is mutated toQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRalanine to reduce aggregation.EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSThe IL2 component is not knownDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS to exhibit receptor bias.PGGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRFc is human IgG1 with N297GMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDmutation to remove glycosylationLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 102) IL2M5IL2 Fc-GGGGS-human IL2 (N88D,DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV Mutein 5 C125A) fusion.SHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHN88D is a mutation of asparagineQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR 88 to aspartic acid. IL2EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSvariants with this mutation haveDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSreduced binding to CD122 andPGGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRtherefore are considered CD25MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRD biased.LISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLTC125A is a mutation of cysteine (SEQ ID NO: 103) 125 alanine to reduceaggregation. Fc is human IgG1 with N297Gmutation to remove glycosylation at this site IL2M6 IL2Fc-(GGGGS)₃-hIL2(H16A, F42A)ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV Mutein 6(Also referred to as Fc- DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV(GGGGS)₃-hIL2(2m)) LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 104) IL2M7 IL2 hIL2(H16A, F42A)-GGGGS-APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYM Mutein 7IgG4st_Fc_holestar PKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVL(Also referred to as hIL2(2m)-ELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSESKGGGGS-IgG4st_Fc_holestar) YGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSFc is from SEQ ID NO: 31 of QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQWO2014/121087, which is an DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEIgG4 with reduced effector EMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSfunction, with addition holeDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNRFTQKSLSLS and star mutationsPGK (SEQ ID NO: 105) IL15M1 IL15 Human IL15-(GGGGS)5-humanNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLEL Mutein 1ILI 5Ra-(GGGGS)4-Fc fusionQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKFc is from SEQ ID NO: 31 of NIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSGGGGSGGGWO2014/121087, which is anGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTEIgG4 with reduced effector CVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSGGGGSGfunction GGGSGGGGSESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 106) IL15M2 IL15 Fc-(GGGGS)₃-human IL15Ra-ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV Mutein 2(GGGGS)₄-human IL15 fusion DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVFc is from SEQ ID NO: 31 ofLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP WO2014/121087, which is anSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL IgG4 with reduced effectorDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS functionLSLGKGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQM FINTS (SEQ ID NO: 107)T1 Targeting Anti-murine PD1 antibodyT1 comprises the T6 comprises the Fab of an moiety 1 anti-PD1 antibodyT2 Targeting CMVpp65(495-503)-GCGGS-NLVPMVATVGCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKS moiety 2(GGGGS)₂-hB2m-(GGGGS)₄- NFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYhHLA.A2 peptide-MHC targetingYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGS moietyGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 108) T3 TargetingHPV16E7(11-19)-GCGGS- YMLDLQPETGCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSmoiety 3 (GGGGS)₂-hB2m-(GGGGS)₄-NFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYhHLA.A2 peptide-MHC targetingYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGS moietyGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 109) T4 TargetingOVA(257-264)-GCGGS- SIINFEKLGCGGSGGGGSGGGGSIQKTPQIQVYSRHPPENGKPNILmoiety 4 (GGGGS)₂-mB2m-(GGGGS)₄-NCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFSKDWSFYILAHTEmH2Kb peptide-MHC targeting FTPTETDTYACRVKHASMAEPKTVYWDRDMGGGGSGGGGSGGmoiety GGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDMELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEPPPSTVSNMA (SEQ ID NO: 110) T5 TargetingMuLV p15E-GCGGS-(GGGGS)₂- KSPWFTTLGCGGSGGGGSGGGGSIQKTPQIQVYSRHPPENGKPmoiety 5 mB2m-(GGGGS)₄-mH2KbNILNCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFSKDWSFYILApeptide-MHC targeting moiety HTEFTPTETDTYACRVKHASMAEPKTVYWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDMELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEPPPSTVSNMA (SEQ ID NO: 111) T6 TargetingAnti LAG3 Antibody T6 comprises a Fab of an anti-LAG3 antibody moiety 6T7 Targeting T3 (i.e., HPV16E7(11-19)-YMLDLQPETGCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKS moiety 7GCGGS-(GGGGS)₂-hB2m- NFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLY(GGGGS)₄-hHLA.A2)-(GGGGS)₄- YTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSFc (knob) GGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPGGGGSGGGGSGGGGSGGGGSESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK (SEQ ID NO: 112)T8 Targeting T2 (i.e., CMVpp65(495-503)-NLVPMVATVGCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKS moiety 8GCGGS-(GGGGS)₂-hB2m- NFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLY(GGGGS)₄-hHLA.A2)-(GGGGS)₄- YTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSFc (knob) GGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPGGGGSGGGGSGGGGSGGGGSESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK (SEQ ID NO: 113)T9 Targeting CMVpp65(495-503)-GCGGS-NLVPMVATVGCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKS moiety 9 (GGGGS)₂-hB2mNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLY (CMVpp65(495-503) in HLA-YTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 114) A*0201) T10 TargetingHPV16E7(11-19)-GCGGS- YMLDLQPETGCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSmoiety 10 (GGGGS)2-hB2m NFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLY(HPV16E7(11-19) in HLA-YTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 115) A*0201)

TABLE 5B Alternate Molecule Name Description Sequence T1-IL2M3Anti-mPD1- IL2 Mutein 3 with anIL2 Mutein 3 was linked to the C-terminal of IL2 N-terminal anti-PD1the heavy chain of an anti-PD1 antibody via a  Mutein 3 antibody(GGGGS)3 linker (SEQ ID NO: 6). T2-IL2M3 CMVpp65_scpMHCMVpp65(495-503)-GCGGS- NLVPMVATVGCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSC-IL2 (GGGGS)₂-hB2m-(GGGGS)₄-NFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLY Mutein 3hHLA.A2-(GGGGS)₄-Fc-hIL2-Rα- YTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGShIL2 GGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFFc is from SEQ ID NO: 31 of VRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVWO2014/121087, which is an DLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDIgG4 with reduced effector GKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTfunction CVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPGGGGSGGGGSGGGGSGGGGSESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK (SEQ ID NO: 116)T2-IL2M6 CMVpp65_scpMH CMVpp65(495-503)-GCGGS-NLVPMVATVGCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKS C-IL2(GGGGS)₂-hB2m-(GGGGS)₄- NFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYMutein 6 hHLA.A2-(GGGGS)₄-Fc-YTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGS (GGGGS)₃-hIL2(H16A, F42A)GGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQF (Also referred to asVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRV CMVpp65(495-503)-GCGGS-DLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYD (GGGGS)₂-hB2m-(GGGGS)₄-GKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGT hHLA.A2-(GGGGS)₄-Fc-CVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALS (GGGGS)₃-hIL2(2m))FYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPS Fc is from SEQ ID NO: 31 ofGQEQRYTCHVQHEGLPKPLTLRWEPGGGGSGGGGSGGGGSGG WO2014/121087, which is anGGSESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC IgG4 with reduced effectorVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV functionLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 117) T3-IL2M3 HPV16E7_scpMHHPV16E7(11-19)-GCGGS- YMLDLQPETGCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKS C-IL2(GGGGS)₂-hB2m-(GGGGS)₄- NFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYMutein 3 hHLA.A2-(GGGGS)₄-Fc-hIL-2Rα-YTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGS hIL2GGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQF Fc is from SEQ ID NO: 31 ofVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRV WO2014/121087, which is anDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYD IgG4 with reduced effectorGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGT functionCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPGGGGSGGGGSGGGGSGGGGSESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGGGGGSGGGGSGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 118) T3-IL2M6HPV16E7_scpMH HPV16E7(11-19)-GCGGS-YMLDLQPETGCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKS C-IL2 (GGGGS)₂-hB2m-(GGGGS)₄- NFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYMutein 6 hHLA.A2-(GGGGS)₄- Fc-YTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGS (GGGGS)₃-hIL2(H16A, F42A)GGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQF (Also referred to asVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRV HPV16E7(11-19)-GCGGS-DLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYD (GGGGS)₂-hB2m-(GGGGS)₄-GKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGT hHLA.A2-(GGGGS)₄-CVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALS Fc-(GGGGS)₃-hIL2(2m))FYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPS Fc is from SEQ ID NO: 31 ofGQEQRYTCHVQHEGLPKPLTLRWEPGGGGSGGGGSGGGGSGG WO2014/121087, which is anGGSESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC IgG4 with reduced effectorVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV functionLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 119) T4-IL2M6 Ova_scpMHC-OVA(257-264)-GCGGS- SIINFEKLGCGGSGGGGSGGGGSIQKTPQIQVYSRHPPENGKPNIL IL2(GGGGS)₂-mB2m-(GGGGS)₄- NCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFSKDWSFYILAHTEMutein 6 mH2Kb-(GGGGS)₄-Fc- FTPTETDTYACRVKHASMAEPKTVYWDRDMGGGGSGGGGSGG(GGGGS)₃-hIL2(H16A, F42A) GGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVR(Also referred to as FDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLOVA(257-264)-GCGGS-(GGGGS)₂-RTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDmB2m-(GGGGS)₄-mH2Kb-(GGGGS)₄-YIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEW Fc-(GGGGS)₃-hIL2(2m))LRRYLKNGNATLLRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADFc is from SEQ ID NO: 31 ofITLTWQLNGEELIQDMELVETRPAGDGTFQKWASVVVPLGKEQYY WO2014/121087, which is anTCHVYHQGLPEPLTLRWEPPPSTVSNMAGGGGSGGGGSGGGG IgG4 with reduced effectorSGGGGSESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPE functionVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 120) T5-IL2M6 MuLVMuLV p15E-GCGGS-(GGGGS)₂- KSPWFTTLGCGGSGGGGSGGGGSIQKTPQIQVYSRHPPENGKPp15E_scpMHC- mB2m-(GGGGS)₄-mH2Kb-NILNCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFSKDWSFYILA IL2 (GGGGS)₄-Fc-(GGGGS)₃-HTEFTPTETDTYACRVKHASMAEPKTVYWDRDMGGGGSGGGGS Mutein 6 hIL2(H16A, F42A)GGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEF (Also referred to as MuLVVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRV p15E-GCGGS-(GGGGS)₂-mB2m-DLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDG (GGGGS)₄-mH2Kb-(GGGGS)₄-CDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCV Fc-(GGGGS)₃-hIL2(2m))EWLRRYLKNGNATLLRTDSPKAHVTHHSRPEDKVTLRCWALGFY Fc is from SEQ ID NO: 31 ofPADITLTWQLNGEELIQDMELVETRPAGDGTFQKWASVVVPLGKE WO2014/121087, which is anQYYTCHVYHQGLPEPLTLRWEPPPSTVSNMAGGGGSGGGGSGG IgG4 with reduced effectorGGSGGGGSESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISR functionTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 121) T6-IL2M3 Anti-mLAG3-IL2 Mutein 3 with an IL2 Mutein 3 was linked to the C-terminal of IL2N-terminal anti-LAG3 the heavy chain of an anti-LAG3 antibody Mutein 3antibody via a (GGGGS)3 linker (SEQ ID NO: 6).

7.1.2. Intracellular Staining of pSTAT5 on Human PBMCs

Cryopreserved human PBMCs were thawed and rested overnight in mediawithout IL-2. The next day, cells were treated with serial dilutions ofdifferent IL-2 variants, then fixed with BD Cytofix™ fixation buffer at37° C. for 12 min. Cells were then permeabilized with pre-chilled BDPhosflow™ Perm Buffer III for 20 min on ice. Cells were then washedtwice with FACS buffer (PBS+2% FBS), followed by incubation with astaining cocktail containing Alexa Fluor® 647-conjugated anti-STAT5pY694 (BD Biosciences) at room temperature for 45 min in the dark. Toidentity different lymphocyte populations in PBMCs, the following panelof antibodies were also included in the staining cocktail:BUV496-anti-CD8, BUV395-anti-CD4, BV421-anti-NKp46, BV711-anti-CD56,BV786-anti-CD3, Alexa Fluor® 488-anti-FoxP3, PE-anti-CD25 (BDBiosciences). Cells were washed twice with FACS buffer before dataacquisition on a BD LSRFortessa™ X-20 flow cytometer. The raw data wereanalyzed using FlowJo v10.

7.1.3. Size-Exclusion Chromatography and Multiangle Light Scattering

Size-exclusion ultra-performance liquid chromatography (SEC) coupledwith multiangle light scattering (MALS) were employed to assess theoligomeric state of different muteins. SEC analysis was conducted on aWaters Acquity UPLC H-Class system. 10 μg of each protein sample wasinjected into a two-column tandem setup consisting of Acquity BEH SECcolumns (200 Å, 1.7 μm, 4.6×150 mm). Flow rate was set at 0.3 ml/min.Mobile phase buffer contains 10 mM sodium phosphate, 500 mM NaCl, pH7.0. UV absorbance at 280 nm, light scattering and refractive indexchanges were monitored using Wyatt Optilab T-Rex and Wyatt-uDawn TreosLS Detector.

7.1.4. FACS Binding Assay

To analyze binding of antibody-IL2 muteins to target antigens on cellsurface, HEK293 cell stably expressing antigen targets were collectedand resuspended in FACS buffer (PBS+2% FBS). For each binding assay,50,000-100,000 cells were incubated with serial dilutions of IL-2muteins in FACS buffer at 4° C. for 30 minutes. Cells were then washedtwice with FACS buffer, and incubated with 1:500 dilution of APC-F(ab)′2anti-mouse IgG Fcγ fragment (Jackson ImmunoResearch Laboratories) for 30minutes at 4° C. At the end of the incubation, cells were washed twicewith FACS buffer and analyzed on BD FACSCelesta™ flow cytometer.

7.1.5. Tumor Inoculation, Treatment and Measurement

A total of 3×10⁵ MC38 cells were injected subcutaneously (s.c.) into theflank of 6-8 weeks old female C57BL/6J mice (Jackson Laboratory). Micewere randomized when tumor size reached 80-100 mm³ and were treatedintraperitoneally with different IL-2/IL-15 agonists. The treatmentswere repeated four more times every other day or semiweekly. Tumors weremeasured semiweekly using a digital caliper and the tumor sizes werecalculated as length×width²/2. In tumor studies where survival wererecorded, loss of survival was defined as death or when tumor reached 20mm in any dimension or 2250 mm³ in total volume.

7.1.6. FACS Analyses of Tumor, Spleen and Blood

Tumors were harvested and processed using gentle MACS™ dissociator(Miltenyi Biotec.) to generate single-cell suspensions. The cells werecounted and then stained with a cocktail of fluorescently-labeledantibodies diluted in BD Horizon Brilliant Buffer.

Spleen and blood were collected from tumor- or non-tumor-bearing miceafter treatment with IL2 muteins. Spleens were mashed through a 70-μmCorning® cell strainer to generate single-cell suspensions. Spleen andblood were then treated with ACK lysing buffer (Lonza) to lyse red bloodcells (RBCs). After RBC lysis, lymphocytes were counted and stained withantibody cocktails diluted in BD Horizon Brilliant Buffer. All stainedsamples were analyzed on a BD LSRFortessa™ X-20 flow cytometer. The rawdata were processed using FlowJo v10.

7.1.7. Antigen-Specific T Cells and Chimeric Antigen Receptor (CAR)T-Cell

Ovalbumin(257-264)-specific mouse OT-1T cells were isolated from thespleen of an OT-1 TCR transgenic mouse (Jackson Laboratory). CMVpp65(495-503)-specific human T cells were expanded from the PBMCs of aCMV+ donor and obtained from ASTARTE Biologics (Cat. #1049). CART cellswere generated by stimulating CD3+ T cells with CD3/CD28 microbeads plus100 U/ml recombinant human IL2 prior to transduction with lentivirus atan MOI=5. The transduced cells were then expanded for 17 days withCD3/CD28 microbeads plus 100 U/ml recombinant human IL2 beforeexchanging media with CTS supplemented OpTmizer™ media (Gibco) lackingIL2. Following an overnight culture at 37° C. and 5% CO₂, a pSTAT5 assaywas performed as described in Section 7.1.2.

7.2. Example 1: Activity of IL-2Rα Attenuated IL2 Muteins In Vitro

To test the ability of IL2 M1 to trigger IL2 receptor signaling invarious immune cell populations, 4×10⁵ human PBMCs were stimulated withincreasing concentrations of IL2M0 or IL2M1 for 20 min at 37° C. As anindicator of IL2 receptor-mediated signaling, levels of intracellularSTAT5 phosphorylation were measured by flow cytometry in immune cellsubsets as described in Section 7.1.2. FIGS. 4A-4C show STAT5 activityin gated Tregs (FIG. 4A), CD8+ T cells (FIG. 4B) and NK cells (FIG. 4C).Compared to IL2M0, IL2 M1 remains equally active on CD8+ T and NK cellswhich lack detectable IL-2Rα expression. However, its activity onIL-2Rα+ Tregs is several orders of magnitude lower than that of IL2-Fc.Therefore, IL2M1 loses the preferentially activity on Tregs over othereffector cell populations in vitro.

7.3. Example 2: Activity of IL-2Rα Attenuated IL2 Muteins on Immune CellPopulations In Vivo

C57BL/6J mice received daily intraperitoneal injection of PBS, 15 μgIL2M0 or IL2M1 for six consecutive days. One day after the lastinjection, spleens were harvested for flow cytometric analysis. Numbersof Tregs, NK cells and CD8+ T cells in the spleens of treated mice areshown in FIGS. 5A-5C, respectively. Relative frequencies of CD8+ andCD4+ T cells within TCRβ+ T cells were quantified (FIG. 5D).

While IL2M0 (IL2-Fc) preferentially expands Tregs in vivo, suchTreg-selectivity was abolished in IL2M1 (a CD122-biased human IL2-Fcfusion). In contrast, IL2M1 induces specific expansion of NK and CD8+ Tcells with minimal effect on the Treg population. Thus, IL2M1 canremodel the peripheral lymphocyte compartment by selectively expandingthe effector cell populations.

7.4. Example 3: Anti-Tumor Activity of IL2 and IL-2Rα Attenuated IL2Muteins as Monotherapies and in Combination with Anti-PD1

C57BL/6J mice were inoculated s.c. with 3×10⁵ MC38 tumor cells on day 0and were randomized on day 8 when average tumor size reached 100 mm³.Mice were then treated intraperitoneally with isotype (10 mg/kg), IL2M0(0.75 mg/kg)+isotype (10 mg/kg), IL2M1 (0.75 mg/kg)+isotype (10 mg/kg),anti-mPD1 (10 mg/kg), IL2M0 (0.75 mg/kg)+anti-mPD1 (10 mg/kg), or IL2M1(0.75 mg/kg)+anti-mPD1 (10 mg/kg) every other day (for IL2M0 and IL2M1)or semi-weekly (for isotype and anti-mPD1) for five total injections.FIG. 6 shows average tumor volumes (mm³+SD) (FIG. 6A) and Kaplan-Meiersurvival curves (FIG. 6B) in each treatment group. Loss of survival wasdefined as death or when tumor reached 20 mm in any dimension or 2250mm³ in total volume.

As a monotherapy, IL2M0 (IL2-Fc) significantly inhibits the progressionof established tumors. Combining it with anti-PD1 furthersynergistically promotes tumor regression and the durability oftumor-free survival. Surprisingly, despite its ability to specificallyexpand NK and CD8+ T cells, the CD122-biased IL2M1 displays littleantitumor effect, even in the presence of anti-PD1.

7.5. Example 4: Dosing Study of IL2M0

C57BL/6J mice were inoculated s.c. with 3×10⁵ MC38 tumor cells on day 0and were randomized on day 8 when average tumor size reached 90 mm³.Mice were then treated intraperitoneally with indicated doses of IL2M0every other day for five total injections. FIG. 7 shows average tumorvolumes (mm³+SD) (FIG. 7A), Kaplan-Meier survival curves (FIG. 7B), andindividual tumor growth curve (7C.1-7C.5). Loss of survival was definedas death or when tumor reached 20 mm in any dimension or 2250 mm³ intotal volume.

This study shows that the anti-tumor efficacy of IL2M0 is highlydependent on the dose injected, with the highest dose being mostefficacious. Effect was observed starting from 5 μg/dose, and increasingthe dose to 10 μg and 20 μg/dose resulted in better tumor control andsurvival benefit.

7.6. Example 5: Anti-Tumor Activity and Toxicity of IL2M1 as Compared toIL2M0

In two different studies, C57BL/6J mice were inoculated s.c. with 3×10⁵MC38 tumor cells on day 0 and were randomized on day 8 when averagetumor size reached 90 mm³ (study #1), or on 7 when average tumor sizereached 70 mm³ (study #2). Mice were then treated intraperitoneally withindicated doses of IL2M0 or IL2 M1 every other day for five totalinjections. Average tumor volumes (mm³+SD) (FIG. 8A-8B, showing resultsof study #1 and study #2, respectively) and percentage of body weightchanges (FIG. 8C, from study #2) for each treatment group were shown.Arrows indicate the days of treatment.

IL2M1 remained ineffective in controlling tumor progression up to 40ug/dose, whereas 10 μg/dose of IL2M0 resulted in significant delay oftumor growth (FIG. 8A). Modest efficacy was observed when IL2M1 wasdosed at 75 or 100 μg/dose (FIG. 8B). However, at these doses, it causedsevere toxicity including significant weight loss (FIG. 8C),hypoactivity and increased mortality rate. It remains to be determinedif the efficacy observed in high dose IL2M1 groups was caused byanti-tumor immunity or tumor malnutrition resulting from the sickness ofthe host mice.

Despite being less effective than the 10 μg/dose of IL2M0 in controllingtumor growth, the 100 μg/dose of IL2M1 did induce massive expansion ofperipheral NK and CD8+ T cells, with a less profound effect on Tregsthan that induced by the 10 μg/dose of IL2M0. However, such massiveexpansion of peripheral effector cell populations did not translate intoa more effective antitumor response.

7.7. Example 6: Anti-Tumor Activity and Toxicity of an IL15 Mutein

IL15 shares the same β/γ receptor subunits with IL2, but it does notbind to IL2-Rα. IL15 is usually expressed as part of an IL15/IL15Rαcomplex on the surface of myeloid cells which is then presented toactivate surrounding lymphocytes (FIG. 1 ). IL15M1, which is comprisesan IL15-IL15Rα fusion, is believed to mimic the trans-presentation ofIL15 by IL15Rα and specifically engage IL-2Rβ/γ, thus triggering similarsignaling as IL2-Rα-attenuated IL2 muteins (e.g., IL2M1).

The anti-tumor activity and toxicity of IL15M1 were tested. C57BL/6Jmice were inoculated s.c. with 3×10⁵ MC38 tumor cells on day 0 and wererandomized on day 7 when average tumor size reached 70 mm³. Mice werethen treated intraperitoneally with indicated doses of IL2M0 and IL15M1every other day for five total injections. FIG. 9 shows average tumorvolumes (mm³+SD) (FIG. 9A) and percentage of body weight changes (FIG.9B) in each treatment group. Arrows indicate the days of treatment. Atday 11, blood from indicated groups were collected and analyzed by flowcytometry, as described in Section 7.1.6. Counts of indicated lymphocytepopulations were quantified (FIGS. 9C.1-C.4).

A modest, yet dose-dependent anti-tumor efficacy was observed afterIL15M1 treatment (FIG. 9A). However, similar to IL2M1, IL15M1 treatmentbrought about concurrent, dose-dependent toxicity in the tumor-bearingmice (FIG. 9B). IL15M1 also induced a dramatic expansion of peripherallymphocytes, mostly NK and CD8+ T cells, but not Tregs in the blood ofthese tumor-bearing mice (FIGS. 9C.1-C.4). Together with the example ofIL2M1, these results indicate that selectively agonizing the IL-2Rβ/γreceptor, whether by a mutant IL2 with abolished IL-2Rα binding or anIL15 mutein, has a suboptimal therapeutic index—limited efficacy inrelation to toxicity. It also suggests that peripheral NK and CD8+ Tcells expanded by IL2M1 and IL15M1 are inefficient in controlling tumorgrowth.

7.8. Example 7: Activity of IL-2Rα Attenuated IL2 Muteins onIntratumoral Lymphocytes

C57BL/6J mice were inoculated s.c. with 3×10⁵ MC38 tumor cells on day 0and were randomized on day 7 when average tumor size reached 100 mm³.Mice were then treated intraperitoneally with PBS, anti-mPD1 (10 mg/kg),IL2M0 (0.75 mg/kg)+anti-mPD1 (10 mg/kg), or IL2M1 (0.75 mg/kg)+anti-mPD1(10 mg/kg) every other day. After three total doses, tumors wereharvested on day 12 to isolate and analyze tumor-infiltratinglymphocytes (TILs). FIGS. 10A.1 to 10A.5 show density tSNE plots oftotal CD45⁺ TILs from each treatment group. Results of quantification ofthe percentage of clusters 1-4 highlighted in FIGS. 10A.1 to 10A.5within total TILs are shown in FIG. 10B. tSNE plots of total TILsoverlaid with the expression of the defining markers for differentlymphocyte populations are shown in FIGS. 10C.1 to 10C.7. These resultsshow that IL2M0 induces a more pronounced expansion of CD8+ T cells inthe tumor than IL2M1.

TCRβ⁺ T cells were further gated out from total TILs. RepresentativeFACS plots and quantification of the frequencies of Ki67⁺IL-2Rα⁺ cellswithin CD8⁺ T cells in the tumor after each treatment are shown in FIG.10D and FIG. 10E, respectively. Results of quantification of thedensities of Ki67⁺IL-2Rα⁺ CD8⁺ T cells in the tumor are shown in FIG.10F. Representative FACS plots and quantification of the frequencies ofFoxP3⁺IL-2Rα⁺ Tregs within CD4⁺ T cells in the tumor are shown in FIG.10G and FIG. 10H. Results of quantification of the densities ofFoxP3⁺IL-2Rα⁺ CD4⁺ Tregs in the tumor are shown in FIG. 10I. Theseresults show that IL2M0, but not IL2M1, increases proliferatingIL-2Rα+CD8+ T cells relative to Tregs in the tumor.

Expression of IL-2Rα and PD1 was evaluated in CD8+ TILs. tSNE plots oftotal CD45+ TILs were overlaid with the expression of these markers, andresults are shown in FIGS. 10J-10L. The expression profiles show asubpopulation of tumor-infiltrating CD8+ T cells that co-express IL-2Rαand PD1. These results show that IL-2Rα and PD1 are co-expressed on someintratumoral CD8+ T cells in the tumor. Given the dynamic expression ofthese two proteins on antigen-specific CD8+ T cells after theiractivation, it is possible that their co-expression occurred on abroader population of CD8+ T cells at time points that were not capturedhere (Kalia et al., 2010, Immunity 32(1): 91-103).

Blood and spleens were harvested from the same tumor-bearing mice forflow cytometric analysis, as described in Section 7.1.6. Results ofquantification of the frequencies NK cells and Tregs in the spleen areshown in FIG. 10M and FIG. 10N. Results of quantification of thefrequencies of NK cells and Tregs in the blood are shown in FIG. 10P andFIG. 10Q. Results of quantification of the relative frequencies of CD8+and CD4+ T cells within TCRβ+ T cells in the spleen are shown in FIG.10O and in the blood are shown in FIG. 10R. These results show thatIL2M1 specifically expands NK and CD8⁺ T cells relative to Tregs,whereas IL2M0 preferentially expands Tregs in the spleen and blood.

The immune cell profiles induced by IL2M0 and IL2M1 are drasticallydifferent in the periphery vs. in the tumor: In the blood and spleen,IL2M0 preferentially expands Treg, whereas IL2M1 causes selectiveexpansion of NK and CD8+ T cells. In the tumor, IL2M0 expands CD8+ Tcells but not Tregs, whereas IL2M1 only slightly increases NK cells.These observations may explain why the massive expansion of peripheralNK and CD8+ T cells induced by IL-2Rα-attenuated IL2 muteins or IL15variants does not translate into good anti-tumor responses, as suchexpansion is not extended to the tumor micro-environment. It alsoexplains why IL2M0, which expands suppressive Tregs in the periphery,can induce effective antitumor immunity. When activated by antigens inthe tumor, tumor-specific T cells upregulate their IL-2Rα, making themmore sensitive to IL2M0 treatment. Together, these results suggest thatpreserving the ability to engage IL-2Rα is required for the anti-tumorefficacy of an IL2-related molecule.

7.9. Example 8: Tumor Tregs are Less Responsive than Splenic Tregs toIL2 Stimulation

Tumor and spleen were harvested from MC38 tumor-bearing C57BL/6J mice toisolate lymphocytes as described in Section 7.1.6. Digested tumor cellswere further subjected to MACS® Cell Separation with CD45 MicroBeads toenrich TILs and. 4×10⁵ TILs or 2×10⁶ splenocytes were stimulated invitro with increasing concentrations of recombinant human IL2 for 20 minat 37° C. FIG. 11 shows percentage of cells that underwent STAT5phosphorylation within gated Tregs (FIG. 11A) and CD8+ T cells (FIG.11B) as determined by FACS according to the assay of Section 7.1.2.

Recombinant human IL2 showed similar EC50 on Tregs from the tumor andspleen. However, while IL2 was able to induce STAT5 phosphorylation inalmost all splenic Tregs, only a fraction of tumor Tregs showed STAT5phosphorylation after IL-2 stimulation. In contrast, CD8+ T cells fromtumor respond to IL-2 treatment with a lower EC50 than those from thespleen. This result suggests tumor Tregs are less responsive thanperipheral Tregs to IL2 stimulation, whereas tumor-infiltrating CD8+ Tcells may be more sensitive to IL2 stimulation than their spleniccounterparts. It also reveals a potential heterogeneity in the tumorTreg compartment.

7.10. Example 9: Activity of IL2M2 and IL2M3 on Lymphocyte Populations

Human PBMCs were stimulated with increasing concentrations of IL2M0,IL2M2 and IL2M3. Level of intracellular STAT5 phosphorylation wasdetermined by FACS as described in Section 7.1.2 and results are shownin FIG. 12A for gated Tregs, FIG. 12B for CD8⁺ T cells, and FIG. 12C forNK cells.

IL2M2 and IL2M3 exhibited similar activities to each other on differentlymphocyte populations. Compared to IL2M0, they showed reducedactivities on all the cell types tested. Overall, they are attenuated onall IL2 receptors while still maintaining selectivity for IL-2Rα+ cells.

C57BL/6J mice were hydrodynamically injected with 5 μg of plasmid DNAencoding IL2M0, IL2M1, IL2M2 or IL2M3 at day 0. Proteins werecontinuously expressed and secreted by hepatocytes to maintain a stableserum concentration. Percentage of body weight changes for eachtreatment group following plasmid injection is shown in FIG. 12D. Incontrast to IL2M0 and IL2M1, IL2M2 and ILM3 injection resulted in nodetectable weight loss, suggestive of markedly reduced toxicity.

7.11. Example 10: Anti-Tumor Activity of IL2M2 Monotherapy in SyngeneicMouse Models

Balbc/J mice were inoculated s.c. with Colon 26 or 4T1 tumor cells onday 0 and were randomized on day 10 or 11 when average tumor sizereached 80 mm³. Right after randomization, mice were hydrodynamicallyinjected with indicated dose of plasmid DNA encoding human Fc or IL2M2under the human ubiquitin C promoter. Proteins were continuouslyexpressed and secreted by hepatocytes to maintain a stable serumconcentration. Average tumor volumes in each treatment group weremeasured. Results (as mm³+SD) for the Colon 26 and 4T1 are shown in FIG.13A and FIG. 13B. Arrows indicate the day of hydrodynamic injection.

C57BL/6J mice were inoculated s.c. with MC38 tumor cells on day 0 andwere randomized on day 7 when average tumor size reached 80 mm³. Micewere then treated intraperitoneally with PBS control, IL2M0 (15 μg), orIL2M2 (50 μg) every three days for four total injections. Average tumorvolumes were measured for each treatment group and results (as mm³+SD)are shown in FIG. 13C.

IL2M2 treatment, either by hydrodynamic delivery of DNA or proteininjection, inhibited tumor progression in multiple syngeneic tumormodels (FIG. 13 ). Despite being ˜100× less potent than IL2M0 onmultiple lymphocyte populations (FIG. 12A-C), a ˜3× higher dose of IL2M2was able to achieve comparable anti-tumor efficacy to IL2M0.

7.12. Example 11: Activity of IL2M4 and IL2M5 on Lymphocyte Populations

Human PBMCs were stimulated with increasing concentrations of IL2M4 (anFc-IL2 fusion protein) or IL2M5. Level of intracellular STAT5phosphorylation were measured by FACS as described in Section 7.1.2.Results are shown in FIG. 14 for gated Tregs (FIG. 14A), CD8⁺ T cells(FIG. 14B) and NK cells (FIG. 14C).

Compared to IL2M4, IL2M5 shows reduced activities on all the cell typestested. However, the degree of attenuation varied among differentlymphocyte populations, with IL-2Rα− NK and CD8+ T cells being moreaffected than the IL-2Rα+ Tregs. Overall, IL2M5 is generally attenuatedbut has increased selectivity for IL-2Rα+ cells.

7.13. Example 12: Anti-Tumor Efficacy of IL2M5 Monotherapy

C57BL/6J mice were inoculated s.c. with MC38 tumor cells on day 0 andwere randomized on day 7 when average tumor size reached 80 mm³. Micewere then treated intraperitoneally with PBS, IL2M4 (15 μg), or IL2M5(100 μg) every three days for four total injections. Average tumorvolumes were measured. The results (in mm³+SD) for each treatment groupare shown in FIG. 15A and for individual tumors in FIGS. 15B to 15D.

Given that IL2M5 is highly attenuated compared to IL2M4, it was used ata higher dose in tumor studies. Despite being >100× less potent thanIL2M4 on multiple lymphocyte populations (FIG. 14A-C), a ˜6× higher doseof IL2M5 achieved a more efficacious tumor control than IL2M4. Fulltumor regression and tumor-free survival was observed in three out offive mice treated with 100 μg/dose of IL2M5.

7.14. Example 13: Oligomerization States of IL-2Rα-Containing Muteins

Size-exclusion ultra-performance liquid chromatography (SEC) coupledwith multiangle light scattering (MALS) were employed to assess theoligomeric state of IL2M2 and IL2M3. IL2M2 contains multiple highmolecular weight species, with the predominant species (˜60%) exhibitinga molar mass consistent with a dimer of dimer (FIG. 16A). In contrast,IL2M3 is less prone to oligomerization. It exists predominantly as adimer (˜77%), along with two minor high molecular weight species (FIG.16B). Arrows indicate the determined molecular weight and relativepercentage of each major population. The equilibrium between differentstructures is illustrated in FIGS. 3A and 3B (for IL2M2 and IL2M3,respectively).

7.15. Example 14: T1-IL2M3 Retains Binding to Cell Surface PD1

The binding of T1-IL2M3, containing an anti-PD1 targeting moiety, wasevaluated by FACS as described in Section 7.1.4. As shown in FIG. 17 ,T1-IL2M3 and the parental anti-mPD1 antibody showed comparable bindingto human HEK293 cell stably expressing mouse PD1 on its surface,suggesting target-binding is minimally affected by fusing the antibodyto IL2M3.

7.16. Example 15: T1-IL2M3 Shows Superior Anti-Tumor Efficacy to theCombination of Anti PD1 & IL2M3

C57BL/6J mice were inoculated s.c. with 3×10⁵ MC38 tumor cells on day 0and were randomized on day 7 when average tumor size reached 100 mm³.Mice were then treated intraperitoneally with isotype (1 mg/kg),anti-mPD1 (1 mg/kg), isotype-IL2M3 (0.5 mg/kg)+anti-mPD1 (0.33 mg/kg),isotype-IL2M3 (1.5 mg/kg)+anti-mPD1 (1 mg/kg), T1-IL2M3 (0.5mg/kg)+isotype (0.33 mg/kg), or T1-IL2M3 (1.5 mg/kg)+isotype (1 mg/kg)semi-weekly for five total injections.

Average tumor volumes were measured. The results (in mm³+SD) for eachtreatment group are shown in FIG. 18A and for individual tumors in FIGS.18B.1 to 18B.4.

While isotype-IL2M3+anti-PD1 was not able to confer tumor control,T1-IL2M3+isotype showed great efficacy at both doses tested, with moremice undergoing complete tumor regression at the higher dose. The amountof IL2M3 moiety delivered in 0.5 mg/kg dose of T1-IL2M3 is very low,equivalent to 5.7 μg/dose of IL2M3. This result reveals the superioranti-tumor efficacy of T1-IL2M3 to the combination of anti-PD1 & IL2M3.It also suggests that PD1-targeting greatly reduced the amount of IL2M3required to achieve efficient tumor control.

Blood was collected from the tumor-bearing mice on day 15 and analyzedby FACS. The frequencies of CD44^(high)CD62^(low) cells and PD1⁺ cellswithin CD8⁺ T cells and FoxP3⁺IL-2Rα⁺ Tregs within CD4⁺ T cells areshown in FIGS. 18C.1-18D.2 and FIGS. 18E.1-18E.2, respectively.

Compared to the non-targeted isotype-IL2M3, T1-IL2M3 induces specificexpansion of activated effector memory CD8+ T cells that areCD44^(hi)CD62L^(lo) and PD1⁺. At the same time, T1-IL2M3 causes lessundesired proliferation of Tregs than isotype-IL2M3. This resultdemonstrates that T1-IL2M3 fusion is able to redirect IL2M3 toantigen-activated CD8+ T cells that express PD1. Activated T cellsupregulate PD1, resulting in T cell inhibition. T1-IL2M3 canspecifically re-activate and expand these cells by stimulating IL2signaling, in addition to blocking PD1 signaling in these cells. Aschematic of the proposed mechanism of action for T1-IL2M3 is shown inFIG. 19 .

7.17. Example 16: Anti-mPD1-IL2 Mutein 3 Fusion Shows SuperiorAnti-Tumor Efficacy to the Combination of Isotype-IL2 Mutein 3 and theParental Anti-PD1 Antibody in Several Murine Syngeneic Tumor Models

The therapeutic efficacy of T1 IL2M3 (an anti-mPD1-IL2 Mutein 3) wasassessed in murine syngeneic tumor models of lung, skin, breast andcolon cancer (using LLC1, B16F10, 4T1, and colon-26 tumor cells,respectively) in two different mouse strains (C57BL/6J and BALB/cJ).

C57BL/6J mice were (a) inoculated s.c. with 2×10⁵ LLC1 tumor cells onday 0 and were randomized on day 7 when average tumor size reached 90mm³, or (b) inoculated s.c. with 3×10⁵ B16F10 tumor cells on day 0 andwere randomized on day 7 when average tumor size reached 80 mm³. Micewere then treated intraperitoneally with isotype (1 mg/kg), isotype-IL 2Mutein 3 (1.5 mg/kg)+anti-mPD1 (1 mg/kg), or anti-mPD1-IL2 Mutein 3 (1.5mg/kg)+isotype (1 mg/kg) semi-weekly for four or five total injections.

BALB/cJ mice were (a) inoculated s.c. with 5×10⁵ 4 T1 tumor cells on day0 and were randomized on day 8 when average tumor size reached 60 mm³,or (b) inoculated s.c. with 1×10⁶ Colon-26 tumor cells on day 0 and wererandomized on day 12 when average tumor size reached 100 mm³. Mice werethen treated intraperitoneally with isotype (1 mg/kg), isotype-IL2Mutein 3 (0.5 mg/kg)+anti-mPD1 (0.33 mg/kg), or anti-mPD1-IL2 Mutein 3(0.5 mg/kg)+isotype (0.33 mg/kg) semi-weekly for five total injections.

Average tumor volumes (mm³+SEM) in each treatment group are shown inFIGS. 20A-20D. Arrows indicate the days of treatment.

In all the models studied, anti-mPD1-IL 2 Mutein 3 displayed betteranti-tumor activity than the combination of isotype-IL2 Mutein 3 andanti-mPD1. Anti-mPD1-IL2 Mutein 3 treatment resulted in complete tumorregression in most of the treated mice in the Colon-26 model (FIG. 20D).In other models (LLC1, B16F10, 4T1) which had been shown to be resistantto general immunotherapies (Mosely et al., 2016, Cancer Immunol Res5(1):29-41), anti-mPD1-IL2 Mutein 3 was able to delay the progression ofestablished tumors (FIGS. 20A-20C).

7.18. Example 17: Anti-mLAG3-IL2 Mutein 3 Fusion Displays BetterAnti-Tumor Efficacy than the Combination of Anti-LAG3+Isotype-IL2 Mutein3

C57BL/6J mice were inoculated s.c. with MC38 tumor cells on day 0 andwere randomized on day 10 when average tumor size reached 50 mm³. Micewere then treated intraperitoneally with isotype (1 mg/kg), anti-mLAG3(1 mg/kg), isotype-IL2 Mutein 3 (0.5 mg/kg)+anti-mLAG3 (0.33 mg/kg),isotype-IL2 Mutein 3 (1.5 mg/kg)+anti-mLAG3 (1 mg/kg), T6-IL2M3(anti-mLAG3-IL2 Mutein 3) (0.5 mg/kg)+isotype (0.33 mg/kg), or T6-IL2M3(anti-mLAG3-IL2 Mutein 3) (1.5 mg/kg)+isotype (1 mg/kg) semi-weekly forfive total injections.

Separately, C57BL/6J mice were inoculated s.c. with MC38 tumor cells onday 0 and were randomized on day 9 when average tumor size reached 80mm³. Mice were then treated intraperitoneally with isotype (0.33 mg/kg),isotype-IL2 Mutein 3 (0.5 mg/kg)+anti-mLAG3 (0.33 mg/kg)+anti-mPD1 (0.33mg/kg), anti-mLAG3-IL2 Mutein 3 (0.5 mg/kg)+isotype (0.33 mg/kg),anti-mLAG3-IL2 Mutein 3 (0.5 mg/kg)+anti-mPD1 (0.33 mg/kg), oranti-mLAG3-IL2 Mutein 3 (0.5 mg/kg)+anti-mPD1 (5 mg/kg) semi-weekly forfour total injections.

Average tumor volumes (mm³+SEM) in each treatment group are shown inFIGS. 21A-21B. Arrows indicate the days of treatment.

The molar amount of IL2M3 moiety delivered by a 0.5 mg/kg dose ofisotype-IL2 Mutein 3 or anti-mLAG3-IL2 Mutein 3 is equivalent to that ina 5.7 μg/kg dose of IL2M2 or IL2M3. While isotype-IL2 Mutein3+anti-mLAG3 was not able to confer tumor control at the studied doses,anti-mLAG3-IL2 Mutein 3+isotype showed augmented anti-tumor efficacy ina dose-dependent manner (FIG. 21A). This result provides another examplethat tumor-responsive T cell-targeting can enhance the antitumorefficacy of IL2M3 by reducing the amount of IL2M3 required to achieve anefficient tumor control.

While anti-mLAG3-IL2M3 alone conferred a partial control of tumorgrowth, its combination with anti-mPD1 resulted in greatly enhancedefficacy, with more mice achieving complete tumor regression (FIG. 21B).This result suggests that combining IL2 Mutein 3 targeted by anon-competing tumor-reactive T cell-targeting antibody with PD1 blockadecan lead to a more robust anti-tumor immunity.

7.19. Example 18: A Peptide MHC-IL2 Mutein Fusion Allows SelectiveStimulation of Antigen-Specific Mouse CD8 T Cells

1.5×10⁶ total splenocytes from an OT-I TCR transgenic mouse (obtainedfrom the Jackson Laboratory) or a control C57BL/6J mouse were stimulatedwith increasing concentrations of T4-IL2M6 or T5-IL2M6 for 20 min at 37°C. Intracellular STAT5 phosphorylation in gated CD8+ T cells wasevaluated by flow cytometry as described in Section 7.1.2.

Results are shown in FIG. 22B. Compared to T5-IL2M6, which targets TCRsagainst an irrelevant antigen, T4-IL2M6 shows two orders of magnitudehigher potency in inducing STAT5 phosphorylation in T4-specific CD8+OT-1T cells, but not on non-specific CD8+ T cells from a control mouse.

The levels of intracellular STAT5 phosphorylation on gated conventionalCD4⁺ T cells and Tregs from the splenocytes of the same mice weremeasured.

Results are shown in FIGS. 23A-23B. Comparable activity was observed forT4-IL2M6 and T5-IL2M6 on both cell types. Unlike CD8⁺ T cells,conventional CD4⁺ T cells and Tregs from the same OT-1 mouse do notexpress the T4-specific OT-1 TCR. Therefore, the selectivity of T4-IL2M6was abolished on these cells.

7.20. Example 19: A pMHC-IL2 Mutein Fusion Allows Selective Stimulationof Antigen-Specific Human CD8⁺ T Cells

1.5×10⁴ CMV pp65-specific human CD8+ T cells were stimulated withincreasing concentrations of T2-IL2M6 or T3-IL2M6 for 20 min at 37° C.Level of intracellular STAT5 phosphorylation was evaluated by flowcytometry as described in Section 7.1.2.

Results are shown in FIG. 24 . Compared to T3-IL2M6, which targets TCRsagainst HPV 16E7 antigen, T2-IL2M6 shows several orders of magnitudehigher potency in inducing STAT5 phosphorylation on these CMVpp65-specific T cells.

7.21. Example 20: Selective CAR-T Expansion by Peptide-MHC Targeted IL2Muteins

7.21.1. Structure of CAR Constructs

Chimeric antigen receptors containing a V_(L)-V_(H) scFv recognizing T3(comprising HLA-A2 loaded with HPV16 E7₁₁₋₁₉ peptide), a huCD8transmembrane domain, a CD137 (4-1BB) co-stimulatory domain, and a CD3ζsignaling domain (as illustrated in FIG. 25A) were constructed using theV_(L) and V_(H) sequences of a single antibody, 17363N. As a CARnegative control, untransduced T cells from the same donor were used.This CAR was cloned into a pLVX lentiviral vector with an EF1a promoterand containing a P2A sequence upstream of the eGFP sequence for trackingCAR-transduced cells. VSV-pseudotyped lentivirus was produced forsubsequent transductions to be carried out using a multiplicity ofinfection (MOI) of 5. Exemplary FACS profiles of cells transduced withthe CAR construct are shown in FIGS. 25B-25C

7.21.2. Generation and Expansion of CAR T Cells

CD3+ T cells from a donor homozygous for HLA-A2 were thawed before beingstimulated at a density of 1.5×10⁶/mL with Dynabeads® Human T-ExpanderCD3/CD28 microbeads plus Aldesleukin (100 IU/mL) for 24 hours.Subsequently, CD3/CD28 microbeads were removed by magnetic separationand activated T cells were then transduced with the CAR construct byspinfection at 2440 rpm for 90 minutes. As a CAR negative control,untransduced T cells from the same donor were used. The transduced anduntransduced cells were then supplemented with Dynabeads® HumanT-Expander CD3/CD28 microbeads (1 bead per 1 T cell) and expanded in CTSsupplemented OpTmizer™ media for 19 days. Cell density was maintainedbetween 1-1.5×10⁶/mL and Aldesleukin was added to the cultures every 48hours and maintained at 100 IU/mL. At the end of expansion, themicrobeads were removed and cells were cryopreserved.

The activities of a variety of peptide-MHC targeted IL2 muteinsillustrated in FIGS. 26A and 26B, containing the attenuated IL2 (H16A,F42A) (also referred to as IL2(2m)), were tested: T7-IL2M7 (which ismonovalent for IL2 and has an HPV peptide-MHC complex), T8-IL2M7 ((whichis monovalent for IL2 and has a CMV peptide-MHC complex), T3-IL2M6(which is bivalent for IL2 and has an HPV peptide-MHC complex), T2-IL2M6(which is monovalent for IL2 and has a CMV peptide-MHC complex). CAR-Tcells were thawed and washed twice in complete RPMI media. Thawed cellswere then rested overnight at 2.8×10⁶/mL in complete RPMI media withoutAldesleukin. After an overnight rest, levels of intracellular STAT5phosphorylation on gated CD4+ or CD8+ populations in these CAR T cellswere evaluated by flow cytometry as described in Section 7.1.2.

7.21.3. Results

Results are shown in FIG. 27A (CD4+ T cells) and 27B (CD8+ T cells). Bygating on CD4+ and CD8+ CAR-T cells engineered to express a CARtargeting and HPV16 E7₁₁₋₁₉ peptide-HLA-A2 complex, the IL2 muteinsT7-IL2M7 and T3-IL2M6 (both of which have a HPV16 E7₁₁₋₁₉ peptide-HLA-A2targeting moiety) show several orders of magnitude higher potency ininducing STAT5 phosphorylation relative to T8-IL2M7 and T2-IL2M6, whichtarget CMV pp65₄₉₅₋₅₀₃-specific T cells.

7.22. Example 21: Selective CAR-T Expansion by Peptide-MHC Targeted IL2Muteins

7.22.1. Generation and Expansion of CAR T Cells

CD3+ T cells from a donor homozygous for HLA-A2 were thawed before beingstimulated with Dynabeads® Human T-Expander CD3/CD28 Microbeads plus3.3×10⁻¹⁰ M recombinant Aldesleukin for 48 hours. Subsequently, CD3/CD28microbeads were removed by magnetic separation and activated T cellswere then transduced with the CAR construct shown in FIG. 27A byspinfection at 2440 rpm for 90 minutes. For a CAR negative control,untransduced T cells from the same donor were used. The transduced anduntransduced cells were then expanded in CTS supplemented OpTmizer™media for 4 days with Dynabeads® Human T-Expander CD3/CD28 microbeads,added at a 1:1 ratio with T cells, plus 3.3×10⁻¹° M Aldesleukin. Theactivated T cells were then harvested, washed two times in OpTmizer™media, and CD3/CD28 microbeads were removed by magnetic separationbefore resting T cells overnight at 37° C. in CTS supplemented OpTmizer™media lacking Aldesleukin. Rested transduced and untransduced T cellswere resuspended and cultured at 1×10⁶/mL in CTS supplemented OpTmizer™media in the presence of Dynabeads® human T-Expander CD3/CD28 microbeads(1 bead to 1 T cell). Each culture was then stimulated with therespective biologics at a concentration of 3.3×10⁻¹⁰ M. Each T cellexpansion culture was supplemented with biologics every 48 hours andmaintained at a density of 1-1.5×10⁶/mL for 17 days. On days 3, 9, 17 ofexpansion, T cell numbers were enumerated and an aliquot of cells wascollected to quantitate changes in the ratio of viable eGFP^(Pos) (CAR+)to eGFP^(Neg) (non-CAR) T cells. Untransduced T cells expanded with eachbiologic were used to define eGFP signal for the CAR-T expansions ateach time-point.

7.22.2. Characterization of CAR-T Cells

At several time-points during the expansion of CAR-T cells, an aliquotof cells was collected to quantitate viable eGFP+ (CAR+) cells by flowcytometry. To identify different T cell populations and expression ofIL2Rα, a combination of the following antibodies were used: BUV395anti-CD4 (BD Biosciences), BUV737 anti-CD4 (BD Biosciences), PE-CY7anti-CD8 (Biolegend), APC-Cy7 anti-CD8 (Biolegend), BV605 anti-CD25 (BDBiosciences). To characterize memory T cell subsets and phenotype, BV421anti-CCR7 (Biolegend), PE-CF594 anti-CD45RO (BD Biosciences),PerCP-Cy5.5 anti-CD45RO (BD Biosciences), BUV395 anti-PD1 (BDBiosciences), and PE-CF594 anti-CD57 (BD Biosciences). Viability wasassessed using 4′,6-diamidino-2-phenylindole (DAPI) (ThermoFisher) orAQUA viability dye (ThermoFisher). All samples were acquired on aBDFortessa™ X-20 or Biorad Ze5 flow cytometer. The raw data wereprocessed using FlowJo v10.

7.22.3. Results & Discussion

To examine the potential of attenuated IL-2 valency in concert withincreasing valency of a given targeting moiety to mediate selectiveCAR-T expansion, selective CAR-T expansion was examined in response to aseries of engineered biologics. In the monovalent format, 1 copy ofattenuated IL2 is combined with 1 copy of the targeting moiety. In thebivalent format, two copies of attenuated IL-2 are combined with twocopies of targeting moiety. To control for non-selective expansion, atargeting moiety not recognized by the CAR-T cells (scHLA-A2 loaded withCMV pp65₄₉₅₋₅₀₃ peptide) was utilized. Structures of the biologicstested are illustrated in FIGS. 26A and 26B.

Results are shown in FIG. 28 . As depicted in FIGS. 28A.1-28A.16, priorto the expansion of transduced T cells in response to scMHC-peptidetargeted IL2 muteins (denoted as Day 0), the frequency of viable CAR-T(eGFP^(Pos)) cells was 38%. By day 3 of expansion, the frequency ofviable CAR-T cells increased to roughly 43% for each culture except forthe bivalent T3-IL2M6 construct where the frequency of CAR decreased to36%. By day 9, the greatest degree of selective CAR-T enrichment isobserved in response to monovalent T7-IL2M7 wherein the percentage ofviable CAR-T cells had increased to 75%. Of note, a less prominentincrease in the percentage of CAR-T was also observed in response to themonovalent T8-IL2M7 even though this biologic does not bind to the CARscFv. While it is unlikely that this expansion resulted from apre-existing T cell repertoire against cytomegalovirus peptide(pp65₄₉₅₋₅₀₃) owing to a lack of an antigen recall response usingmatched donor PBMC (as per the vendor), this possibility cannot be fullyruled out. Between day 9 and 17 of expansion, the percentage of CAR inresponse to the monovalent T7-IL2M7 and T8-IL2M7 remained similar. Aslight increase in the frequency of CAR-T was observed in response toIL2 (Aldesleukin) and bivalent T3-IL2M6, whereas no further change wasobserved in response to bivalent T2-IL2M6.

To evaluate the potential of biologics that concomitantly bind to thescFv of a CAR and deliver attenuated IL2 signals to mediate selectiveexpansion of CAR-T, the total number of viable T cells against the ratioof CAR-T cells (EGFP^(Pos)) to non-CAR-T cells EGFP^(Neg)) was plotted.As shown in FIGS. 28B.1-B.4, selective enrichment of CAR-T occurredbetween day 3 and day 9 in response to T7-IL2M7 (open circle) as theratio of eGFP^(Pos)/eGFP^(Neg) increased from 0.73 to 3.12. In addition,the number of T cells more than doubled. By day 17, at culturetermination, the greatest number of expanded total T cells was observedin response to Aldesleukin (black square) followed by monovalentT7-IL2M7 (open circle); however, the ratio of CAR^(Pos) to CAR^(Neg) Tcells was maximal in cultures expanded with T7-IL2M7 indicating that aselective and targeted expansion of CAR-T had occurred. As shown in FIG.28C, the absolute number of viable CAR-T cells for each expansioncondition are plotted as a function of time in culture. While IL-2(Aldesleukin) yielded the greatest number of total T cells (FIG. 28B.4),due to selective CAR-T expansion, monovalent T7-IL2M7 generated themaximal number of viable CAR-T cells (FIG. 28C). Minimal expansion ofCAR-T occurred in response to monovalent T8-IL2M7 demonstrating theimportance of the targeting moiety. Of note, CAR-T expansion was low inresponse to bivalent T3-IL2M6, potentially indicating over-activation ofthe CAR-T cells resulting in cell death. Alternatively, the bivalentT3-IL2M6 may cross-link separate CAR-T cells resulting in CAR-Tfratricide. These studies show that the monovalent format structure issuperior for mediating selective CAR-T expansion.

7.23. Example 22: In Vivo Pharmacokinetic Assessment of Peptide-MHCTargeted IL2 Muteins

7.23.1. Methods

To assess the pharmacokinetics of indicated IL2 muteins in plasma, bothimmunocompetent C57BL/6J and immunodeficient NSG (NOD-scid IL2Rgnull)mice were intraperitoneally injected with a single dose of 12 μg of eachprotein. Blood samples were collected at 2, 24, 48 and 72 hr afterdosing followed by centrifugation for 10 min at 10,000 RPM to isolateplasma. 1 μl of each plasma sample were analyzed by SDS-PAGE followed byimmunoblotting with antibodies against human IgG Fc. 2 ng of eachpurified protein (spiked into 1 μl naïve plasma) were loaded onto thesame gels to help estimate the absolute levels of each protein in theplasma samples.

7.23.2. Results

Results are shown in FIG. 29 . Compared to IL2M0 (referred to in FIG. 29as IL2-Fc), the targeted IL2 muteins shown in FIGS. 26A and 26Bdemonstrated improved PK in an avidity dependent manner. In addition, incomparison to IL2-Fc, both bivalent and monovalent constructs showdelayed clearance in circulation, with the monovalent constructs(T7-IL2M7 and T8-IL2M7) being longest-lasting. This result suggests IL2attenuation can significantly reduce its receptor-mediated clearance inan avidity-dependent manner, and therefore increases the chance for atargeted attenuated IL2 mutein to reach target cells. Despite the moredurable presence in circulation, bivalent T2-IL2M6 still shows a fasterelimination from the blood than the targeting moiety alone (T2-Fc),suggesting that clearance of T2-IL2M6 is mainly driven by IL2M6.

8. SPECIFIC EMBODIMENTS, CITATION OF REFERENCES

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the disclosure(s). The presentdisclosure is exemplified by the numbered embodiments set forth below.

In preferred aspects of the numbered embodiments below and the claimswhich follow, the IL2 domains, the IL2 receptors, Fc domains, MHCdomains, β2M, and the variants thereof preferably comprise the aminoacid sequences of human IL2, human IL2 receptors, human Fc domains,human MHC domains, human β2M, and variants thereof, for example variantswith at least about 90%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99% or 100% sequenceidentity to such human sequence.

1. An IL2 agonist comprising:

-   -   (a) an IL2 moiety comprising an IL2 domain;    -   (b) optionally, a multimerization moiety;    -   (c) optionally, a targeting moiety; and    -   (d) optionally, a stabilization moiety.

2. The IL2 agonist of embodiment 1, which comprises an IL2 mutein havingthe configuration of:

-   -   (a) IL2M0;    -   (b) IL2M1;    -   (c) IL2M2;    -   (d) IL2M3;    -   (e) IL2M4;    -   (f) IL2M5;    -   (g) IL2M6; or    -   (h) IL2M7.

3. The IL2 agonist of embodiment 1 or embodiment 2, wherein the IL2moiety is an IL2-Rα-biased IL2 mutein.

4. The IL2 agonist of any one of embodiments 1 to 3, wherein the IL2moiety and/or the IL2 agonist has 50-fold to 1,000-fold attenuatedbinding to human IL2-Rβ as compared to wild type IL2.

5. The IL2 agonist of any one of embodiments 1 to 4, wherein the IL2moiety and/or the IL2 agonist has up to 100-fold, up to 500-fold, up to1,000-fold or up to 5,000-fold attenuated binding to human IL2-Rα ascompared to wild-type human IL2.

6. The IL2 agonist of any one of embodiments 1 to 5, wherein the ratioof the binding affinity of IL2 agonist to the high affinity IL2 receptorto the binding affinity of the IL2 agonist to the intermediate affinityIL2 receptor is equal to or greater than the corresponding ratio forwild type IL2.

7. The IL2 agonist of any one of embodiments 1 to 6, wherein the IL2moiety and/or the IL2 agonist has an E50 for the high affinity IL2receptor that is 100-fold to 10,000-fold lower than the E50 for theintermediate affinity IL2 receptor.

8. The IL2 agonist of embodiment 1, wherein the IL2 moiety is anIL2-Rβ-biased IL2 mutein.

9. The IL2 agonist of embodiment 1 or embodiment 8, wherein the IL2moiety and/or the IL2 agonist has (a) 50-fold to 1,000-fold or (b)50-fold to 5,000-fold attenuated binding to human IL2-Rα as compared towild type IL2.

10. The IL2 agonist of any one of embodiments 1, 8 and 9, wherein theIL2 moiety and/or the IL2 agonist has up to 50-fold attenuated bindingto human IL2-Rβ as compared to wild-type human IL2.

11. The IL2 agonist of any one of embodiments 1 and 8 to 10, wherein theratio of the binding affinity of the IL2 moiety and/or the IL2 agonistto the intermediate affinity IL2 receptor to the binding affinity of theIL2 moiety and/or the IL2 agonist to the high affinity IL2 receptor isequal to or greater than the corresponding ratio for wild type IL2.

12. The IL2 agonist of any one of embodiments 1 and 7 to 11, wherein theIL2 moiety and/or the IL2 agonist has an E50 for the high affinity IL2receptor that is 10-fold to 100-fold lower than the E50 for theintermediate affinity IL2 receptor.

13. The IL2 agonist of any one of embodiments 1 to 12, wherein the IL2moiety and/or the IL2 agonist has attenuated binding affinity to thehigh affinity IL2 receptor compared to wild type IL2.

14. The IL2 agonist of embodiment 13, wherein the binding affinity isattenuated by up to 1,000-fold or up to 5,000 fold, or wherein thebinding affinity is attenuated by:

-   -   (a) 10-fold to 1,000-fold;    -   (b) 50-fold to 5,000-fold;    -   (c) up to 10-fold;    -   (d) up to 50-fold;    -   (e) up to 100-fold; or    -   (f) up to 200-fold.

15. The IL2 agonist of any one of embodiments 1 to 14, wherein the IL2moiety and/or the IL2 agonist has higher cytokine activity on tumorreactive lymphocytes than on peripheral lymphocytes.

16. The IL2 agonist of embodiment 15, wherein the IL2 moiety and/or theIL2 agonist has at least 5-fold or at least 10-fold higher cytokineactivity on tumor reactive lymphocytes than on peripheral lymphocytes.

17. The IL2 agonist of any one of embodiments 1 to 16, which has atherapeutic index of at least 1.

18. The IL2 agonist of embodiment 17, which has a therapeutic index ofat least 2.

19. The IL2 agonist of embodiment 17, which has a therapeutic index ofat least 5.

20. The IL2 agonist of embodiment 17, which has a therapeutic index ofat least 10.

21. The IL2 agonist of embodiment 17, which has a therapeutic index ofat least 50.

22. The IL2 agonist of any one of embodiments 17 to 21, which has atherapeutic index of up to 500.

23. The IL2 agonist of any one of embodiments 17 to 21, which has atherapeutic index of up to 250.

24. The IL2 agonist of any one of embodiments 1 to 23, which has atherapeutic index of about 2.

25. The IL2 agonist of any one of embodiments 1 to 23, which has atherapeutic index of about 10.

26. The IL2 agonist of any one of embodiments 1 to 23, which has atherapeutic index of about 20.

27. The IL2 agonist of any one of embodiments 1 to 23, which has atherapeutic index of about 50.

28. The IL2 agonist of any one of embodiments 1 to 23, which has atherapeutic index of about 100.

29. The IL2 agonist of any one of embodiments 1 to 23, which has atherapeutic index of about 200.

30. The IL2 agonist of any one of embodiments 1 to 29, wherein the IL2moiety and/or the IL2 agonist is not pegylated.

31. The IL2 agonist of any one of embodiments 1 to 30, wherein the IL2moiety and/or the IL2 agonist does not comprise a cytokine other thanIL2.

32. The IL2 agonist of any one of embodiments 1 to 31, wherein the IL2moiety and/or the IL2 agonist does not contain an anti-IL2 antibody orantibody fragment.

33. The IL2 agonist of any one of embodiments 1 to 32, wherein the IL2moiety and/or the IL2 agonist does not contain an anti-DNA antibody orantibody fragment.

34. The IL2 agonist of any one of embodiments 1 to 33, wherein the IL2domain does not comprise a substitution at the position D20 as comparedto human IL2.

35. The IL2 agonist of any one of embodiments 1 to 34, wherein the IL2domain does not comprise a substitution at the position Q126 as comparedto human IL2.

36. The IL2 agonist of any one of embodiments 1 to 35, wherein the IL2moiety and/or the IL2 agonist does not contain a non-binding variabledomain.

37. The IL2 agonist of any one of embodiments 1 to 36, wherein the IL2moiety comprises an IL2 domain comprising an amino acid sequence havingat least about 90% sequence identity to mature human IL2.

38. The IL2 agonist of any one of embodiments 1 to 36, wherein the IL2moiety comprises an IL2 domain comprising an amino acid sequence havingat least about 93% sequence identity to mature human IL2.

39. The IL2 agonist of any one of embodiments 1 to 36, wherein the IL2moiety comprises an IL2 domain comprising an amino acid sequence havingat least about 96% sequence identity to mature human IL2.

40. The IL2 agonist of any one of embodiments 1 to 39, wherein the IL2moiety comprises an IL2 variant which has the amino acid substitutionC125S, C125A or C125V.

41. The IL2 agonist of any one of embodiments 1 to 40, wherein the IL2domain comprises an IL2 sequence that has one or more substitutions toreduce O-linked glycosylation.

42. The IL2 agonist of embodiment 41, wherein the IL2 domain has asubstitution at a position corresponding to residue 3 of human IL2.

43. The IL2 agonist of embodiment 42, wherein the amino acidsubstitution is T3A, T3G, T3Q, T3E, T3N, T3D, T3R, or T3K.

44. The IL2 agonist of any one of embodiments 1 to 43, wherein the IL2domain has a substitution at a position corresponding to methionine 104of human IL2 with a neutral amino acid.

45. The IL2 agonist of embodiment 44, wherein the neutral amino acid isalanine.

46. The IL2 agonist of any one of embodiments 1 to 45, wherein the IL2domain is a full-length human IL2 domain.

47. The IL2 agonist of any one of embodiments 1 to 45, wherein the IL2domain has an N-terminal alanine deletion as compared to full lengthmature human IL2.

48. The IL2 agonist of any one of embodiments 1 to 47, wherein the IL2domain comprises an IL2 variant which has an amino acid substitution atposition N88.

49. The IL2 agonist of embodiment 48, wherein the amino acidsubstitution is N88D.

50. The IL2 agonist of any one of embodiments 1 to 47, wherein the IL2domain comprises an IL2 variant which has amino acid substitutions atpositions H16 and F42.

51. The IL2 agonist of embodiment 50, wherein the amino acidsubstitutions are H16A and F42A.

52. The IL2 agonist of any one of embodiments 1 to 51, wherein the IL2moiety comprises an IL-2Rα domain comprising an IL2 binding portion ofIL-2Rα fused to the IL2 domain.

53. The IL2 agonist of embodiment 52, wherein the IL2-Rα domain or theIL2 binding portion of IL-2Rα is the extracellular domain of IL-2Rα oran IL2 binding portion thereof.

54. The IL2 agonist of embodiment 52 or embodiment 53, wherein theIL2-Rα domain or the IL2 binding portion comprises or consists of anamino acid sequence having at least about 90%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about99% or 100% sequence identity to an IL2 binding portion of human IL-2Rα,optionally wherein said binding portion has:

-   -   (a) at least 160 amino acids, at least 161 amino acids, at least        162 amino acids, at least 164 amino acids or at least 165 amino        acids of human IL2-Rα; and/or    -   (b) up to 251, up to 240, up to 230, up to 220, up to 210, up to        200, up to 190, up to 180 or up to 170 amino acids of the        extracellular domain of human IL2-Rα.

55. The IL2 agonist of any one of embodiments 52 to 54, wherein theIL2-Rα domain or the IL2 binding portion has an amino acid sequence withat least about 90%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, at least about 99% or 100% sequenceidentity to amino acids 22-186 of IL-2Rα, amino acids 22-240 of IL-2Rα,and/or amino acids 22-272 of IL-2Rα.

56. The IL2 agonist of any one of embodiments 52 to 55, wherein theIL2-Rα domain or the IL2 binding portion comprises or consists of anamino acid sequence having at least about 90%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about99% or 100% sequence identity to amino acids 22-186, with or without anadditional up to 5 amino acids, up to 10 amino acids, up to 15 aminoacids, up to 20 amino acids, up to 30 amino acids, or up to 40 aminoacids C-terminal to amino acid residue 186, of human IL2-Rα.

57. The IL2 agonist of any one of embodiments 52 to 56, wherein theIL-2Rα domain is N-terminal to the IL2 domain.

58. The IL2 agonist any one of embodiments 52 to 56, wherein the IL-2Rαdomain is C-terminal to the IL2 domain.

59. The IL2 agonist of any one of embodiments 52 to 58, wherein the IL2domain and the IL2-Rα domain are connected via a linker.

60. The IL2 agonist of embodiment 59, wherein the linker is or comprisesa glycine-serine linker.

61. The IL2 agonist of embodiment 60, wherein the linker comprises theamino acid sequence G₄S (SEQ ID NO:57).

62. The IL2 agonist of embodiment 61, wherein the linker is or comprisesa multimer of the amino acid sequence G₄S (SEQ ID NO:57).

63. The IL2 agonist of embodiment 62, wherein the multimer comprises 2,3, 4, 5 or 6 repeats of the amino acid sequence G₄S (SEQ ID NO:57).

64. The IL2 agonist of any one of embodiments 1 to 63, which comprises amultimerization and/or a stabilization moiety.

65. The IL2 agonist of embodiment 64, wherein the multimerization moietyand/or the stabilization moiety is or comprises an Fc domain.

66. The IL2 agonist of embodiment 65, wherein the Fc domain is an IgG1,IgG2, IgG3 or IgG4 Fc domain.

67. The IL2 agonist of embodiment 65 or embodiment 66, wherein the Fcdomain has reduced effector function.

68. The IL2 agonist of embodiment 67, wherein the Fc domain comprisesthe amino acid sequence of SEQ ID NO:31 of WO2014/121087 or a portionthereof, e.g., as set forth in Table 4 and/or Section 6.5.1.1.

69. The IL2 agonist of any one of embodiments 1 to 68, which comprises astabilization moiety.

70. The IL2 agonist of embodiment 69, wherein the stabilization moietyis human serum albumin, a human serum albumin binder, an XTEN, a PAS, acarbohydrate, a polysialic acid, a hydrophilic polymer, a fatty acid, oran Fc domain.

71. The IL2 agonist of embodiment 70, wherein the stabilization moietyis a human serum albumin binder.

72. The IL2 agonist of embodiment 71, wherein the human serum albuminbinder is Adnectin PKE, AlbudAb, or an albumin binding domain.

73. The IL2 agonist of embodiment 72, wherein the stabilization moietyis an Fc domain.

74. The IL2 agonist of embodiment 73, wherein the Fc domain is amonomeric Fc domain.

75. The IL2 agonist of embodiment 73 or embodiment 74, wherein Fc domainhas reduced effector function.

76. The IL2 agonist of embodiment 70, wherein the stabilization moietyis a hydrophilic polymer.

77. The IL2 agonist of embodiment 76, wherein the hydrophilic polymer isPEG.

78. The IL2 agonist of embodiment 77, wherein the PEG has a molecularweight ranging from about 7.5 kDa to about 80 kDa.

79. The IL2 agonist of embodiment 78, wherein the PEG has a molecularweight ranging from about 30 kDa to about 60 kDa, optionally wherein themolecular weight is about 50 kDa.

80. The IL2 agonist of any one of embodiments 76 to 79, wherein thehydrophilic molecule is attached to the IL-2Rβ binding surface of IL2.

81. The IL2 agonist of any one of embodiments 1 to 80 which is a dimer.

82. The IL2 agonist of embodiment 81, which is a homodimer.

83. The IL2 agonist of embodiment 81, which is a heterodimer.

84. The IL2 agonist of any one of embodiments 1 to 80 which is amonomer.

85. The IL2 agonist of any one of embodiments 1 to 84, which ismonovalent for the IL2 moiety.

86. The IL2 agonist of any one of embodiments 1 to 85, which is bivalentfor the IL2 moiety.

87. The IL2 agonist of any one of embodiments 1 to 86, which is orcomprises an Orientation 1 IL2 agonist.

88. The IL2 agonist of any one of embodiments 1 to 87, which comprises:

-   -   (a) An IL2 moiety comprising:        -   (i) An IL2 or an IL2 variant (e.g., IL2 N88D) domain, with            or without a substitution at C125 that reduces aggregation            (e.g., C125S or C125A or C125V);        -   (ii) A linker (e.g., as described in Section 6.7), e.g., a            linker comprising 10 or more amino acids and/or comprising            or consisting of (G₄S)_(n) (SEQ ID NO:57), optionally            wherein n≥2, e.g., wherein N is 3, 4, 5 or greater; and        -   (iii) IL2 binding portion of IL-2Rα;    -   (b) A linker (e.g., as described in Section 6.7), e.g., a linker        comprising or consisting of (G₄S)_(n) (SEQ ID NO:57), optionally        wherein n 1, e.g., wherein N is 1, 2, 3, 4, 5 or greater; and    -   (c) An Fc domain (e.g., IgG1 or IgG4, with or without        substitutions that reduce glycosylation and/or effector function        as described in Section 6.5.1 and subsections thereof).

89. The IL2 agonist of any one of embodiments 1 to 86, which is orcomprises an Orientation 2 IL2 agonist.

90. The IL2 agonist of any one of embodiments 1 to 89, which comprises:

-   -   (a) An Fc domain (e.g., IgG1 or IgG4, with or without        substitutions that reduce glycosylation and/or effector function        as described in Section 6.5.1 and subsections thereof);    -   (b) A linker (e.g., as described in Section 6.7), e.g., a linker        comprising or consisting of (G₄S)_(n) (SEQ ID NO:57), optionally        wherein n 1, e.g., wherein N is 1, 2, 3, 4, 5 or greater; and    -   (c) An IL2 or an IL2 variant (e.g., IL2 N88D) domain, with or        without a substitution at C125 that reduces aggregation (e.g.,        C125S or C125A or C125V).

91. The IL2 agonist of any one of embodiments 1 to 89, which comprises:

-   -   (a) An Fc domain (e.g., IgG1 or IgG4, with or without        substitutions that reduce glycosylation and/or effector function        as described in Section 6.5.1 and subsections thereof);    -   (b) A linker (e.g., as described in Section 6.7), e.g., a linker        comprising or consisting of (G₄S)_(n) (SEQ ID NO:57), optionally        wherein n 1, e.g., wherein N is 1, 2, 3, 4, 5 or greater; and    -   (c) An IL2 moiety comprising:        -   (i) An IL2 or an IL2 variant (e.g., IL2 N88D) domain, with            or without a substitution at C125 that reduces aggregation            (e.g., C125S or C125A or C125V);        -   (ii) A linker (e.g., as described in Section 6.7), e.g., a            linker comprising 10 or more amino acids and/or comprising            or consisting of (G₄S)_(n) (SEQ ID NO:57), optionally            wherein n 2, e.g., wherein N is 3, 4, 5 or greater;            and (iii) An IL2 binding portion of IL2Rα.

92. The IL2 agonist of any one of embodiments 1 to 86, which is orcomprises an Orientation 3 IL2 agonist.

93. The IL2 agonist of any one of embodiments 1 to 92, which comprises:

-   -   (a) An scFv or a heavy chain variable region of a Fab        (associated with a corresponding light chain variable region on        a separate polypeptide) (e.g., as described in Section 6.4.2 and        subsections thereof);    -   (b) A linker (e.g., as described in Section 6.7), e.g., a linker        comprising or consisting of (G₄S)_(n) (SEQ ID NO:57), optionally        wherein n 1, e.g., wherein N is 1, 2, 3, 4, 5 or greater;    -   (c) An Fc domain (e.g., IgG1 or IgG4, with or without        substitutions that reduce glycosylation and/or effector function        as described in Section 6.5.1 and subsections thereof);    -   (d) A linker (e.g., as described in Section 6.7), e.g., a linker        comprising or consisting of (G₄S)_(n) (SEQ ID NO:57), optionally        wherein n 1, e.g., wherein N is 1, 2, 3, 4, 5 or greater; and    -   (e) An IL2 moiety comprising:        -   (i) IL2 or an IL2 variant (e.g., IL2 N88D) domain, with or            without a substitution at C125 that reduces aggregation            (e.g., C125S, C125A or C125V);        -   (ii) A linker (e.g., as described in Section 6.7), e.g., a            linker comprising 10 or more amino acids and/or comprising            or consisting of (G₄S)_(n) (SEQ ID NO:57), optionally            wherein n 2, e.g., wherein N is 3, 4, 5 or greater; and        -   (iii) An IL2 binding portion of IL-2Rα (e.g., as described            in Section 6.3).

94. The IL2 agonist of any one of embodiments 1 to 92, which comprises:

-   -   (a) A peptide-MHC complex (e.g., as described in Section 6.4.3)        comprising:        -   (i) An MHC peptide;        -   (ii) A linker (e.g., as described in Section 6.7 or            subsections thereof, for example in Section 6.7.1);        -   (iii) Optionally, a β2-microglobulin (β2m) domain;        -   (iv) Optionally, a linker (e.g., as described in Section 6.7            or subsections thereof, for example in Section 6.7.1); and        -   (v) MHC;    -   (b) Optionally, a linker (e.g., as described in Section 6.7),        e.g., a linker comprising or consisting of (G₄S)_(n) (SEQ ID        NO:57), optionally wherein n 1, e.g., wherein N is 1, 2, 3, 4, 5        or greater;    -   (c) An Fc domain (e.g., IgG1 or IgG4, with or without        substitutions that reduce glycosylation and/or effector function        as described in Section 6.5.1 and subsections thereof);    -   (d) A linker (e.g., as described in Section 6.7), e.g., a linker        comprising or consisting of (G₄S)_(n) (SEQ ID NO:57), optionally        wherein n 1, e.g., wherein N is 1, 2, 3, 4, 5 or greater;    -   (e) An IL2 moiety comprising:        -   (i) An IL2 or IL2 variant (e.g., IL2 N88D) domain, with or            without a substitution at C125 that reduces aggregation            (e.g., C125S, C125A or C125V);        -   (ii) A linker (e.g., as described in Section 6.7), e.g., a            linker comprising 10 or more amino acids and/or comprising            or consisting of (G₄S)_(n) (SEQ ID NO:57), optionally            wherein n 2, e.g., wherein N is 3, 4, 5 or greater; and        -   (iii) An IL2 binding portion of IL-2Rα (e.g., as described            in Section 6.3).

95. The IL2 agonist of any one of embodiments 1 to 86, which is orcomprises an Orientation 4 IL2 agonist.

96. The IL2 agonist of any one of embodiments 1 to 86 and 95, whichcomprises:

-   -   (a) A first polypeptide comprising:        -   (i) A targeting moiety, e.g., peptide-MHC complex (e.g., as            described in Section 6.4.3), Fab domain (e.g., as described            in Section 6.4.2.2) (e.g., a heavy chain of a Fab associated            with a third polypeptide comprising the light chain of the            Fab), or scFv domain (e.g., as described in Section            6.4.2.1);        -   (ii) An optional linker (e.g., as described in Section 6.7);            and        -   (iii) A first Fc domain; and    -   (b) A second polypeptide comprising:        -   (i) IL2 moiety comprising an IL2 or IL2 variant domain            (e.g., as described in Section 6.3 (e.g., an IL2 domain with            the substitutions H16A, F42A, also referred to as IL2(2m)),            with or without a substitution at C125 that reduces            aggregation (e.g., C125S, C125A or C125V);        -   (ii) An optional linker (e.g., as described in Section 6.7);            and        -   (iii) A second Fc domain that can dimerize (e.g.,            heterodimerize) with the first Fc domain (e.g., as described            in Section 6.5.1.2).

97. The IL2 agonist of any one of embodiments 1 to 86, 95 and 96, whichcomprises:

-   -   (a) A first polypeptide, comprising:        -   (i) A peptide-MHC complex (e.g., as described in Section            6.4.3) comprising:            -   (1) An MHC peptide;            -   (2) A linker (e.g., as described in Section 6.7 or                subsections thereof, for example in Section 6.7.1);            -   (3) Optionally, a β2-microglobulin (β2m) domain;            -   (4) Optionally, a linker (e.g., as described in Section                6.7 or subsections thereof, for example in Section                6.7.1); and            -   (5) An MHC;        -   (ii) Optionally, a linker (e.g., as described in Section            6.7); and        -   (iii) A first Fc domain.    -   (b) A second polypeptide, comprising:        -   (i) An IL2 moiety comprising an IL2 or IL2 variant (e.g.,            IL2 H16A, F42A) domain, with or without a substitution at            C125 that reduces aggregation (e.g., C125S, C125A or C125V);        -   (ii) Optionally, a linker (e.g., as described in Section            6.7); and        -   (iii) A second Fc domain that is not identical to, but can            heterodimerize with, the first Fc domain (e.g., as described            in Section 6.5.1.2).

98. The IL2 agonist of any one of embodiments 1, 87 and 88, which (a)has or comprises an amino acid sequence having the configuration of or(b) comprises the amino acid sequence of IL2M0.

99. The IL2 agonist of any one of embodiments 1, 87 and 88, which (a)has or comprises an amino acid sequence having the configuration of(e.g., IL2 moiety—optional linker—IL2Rα-optional linker—Fc domain) or(b) comprises the amino acid sequence of IL2M2.

100. The IL2 agonist of any one of embodiments 1 and 91 to 94, which (a)has or comprises an amino acid sequence having the configuration of(e.g., IL2Rα-optional linker—IL2 moiety—optional linker—Fc domain) or(b) comprises the amino acid sequence of IL2M3.

101. The IL2 agonist of any one of embodiments 1, 89 and 90, which (a)has or comprises an amino acid sequence having the configuration of(e.g., Fc domain-optional linker—IL2 moiety) or (b) comprises the aminoacid sequence of IL2M4.

102. The IL2 agonist of any one of embodiments 1, 89 and 90, which (a)has or comprises an amino acid sequence having the configuration of(e.g., Fc domain-optional linker—IL2 moiety) or (b) comprises the aminoacid sequence of IL2M5.

103. The IL2 agonist of any one of embodiments 1 and 95 to 97, which (a)has or comprises an amino acid sequence having the configuration of(e.g., Fc domain-optional linker—IL2 moiety) or (b) comprises the aminoacid sequence of IL2M6.

104. The IL2 agonist of any one of embodiments 1 and 95 to 97, which (a)has an amino acid sequence having the configuration (e.g., IL2moiety—optional linker—Fc domain) of IL2M7 or (b) comprises the aminoacid sequence of IL2M7, in each case optionally associated with apolypeptide chain comprising a targeting moiety-optional linker-Fcdomain, e.g., a polypeptide chain comprising the sequence orconfiguration of T7 or T8.

105. An IL2 agonist comprising:

-   -   (a) an IL2 moiety comprising an IL2 domain and an IL2 binding        portion of IL2-Rα; and    -   (b) an Fc domain.

106. The IL2 agonist of embodiment 105, wherein the IL2 binding portionof IL2-Rα is N-terminal to the IL2 domain.

107. The IL2 agonist of embodiment 105, wherein the IL2 binding portionof IL2-Rα is C-terminal to the IL2 domain.

108. The IL2 agonist of any one of embodiments 105 to 107, wherein theFc domain is N-terminal to the IL2 moiety.

109. The IL2 agonist of any one of embodiments 105 to 107, wherein theFc domain is C-terminal to the IL2 moiety.

110. The IL2 agonist of any one of embodiments 105 to 109, wherein theIL2 domain and IL2 binding portion of IL2-Rα are connected via a linker,optionally wherein the linker comprises 10 or more or 15 or more aminoacids.

111. The IL2 agonist of embodiment 110, wherein the linker is orcomprises G₄S (SEQ ID NO:57) or a multimer thereof.

112. The IL2 agonist of embodiment 111, wherein the linker comprises asingle G₄S (SEQ ID NO:57).

113. The IL2 agonist of embodiment 111, wherein the linker comprisestwo, three, four or five repeats of G₄S (SEQ ID NO:57).

114. The IL2 agonist of any one of embodiments 105 to 113, wherein theIL2 moiety and the Fc domain are connected via a linker, optionallywherein the linker comprises 5 or more or 10 or more amino acids.

115. The IL2 agonist of embodiment 114, wherein the linker is orcomprises G₄S (SEQ ID NO:57) or a multimer thereof.

116. The IL2 agonist of embodiment 115, wherein the linker comprises asingle G₄S (SEQ ID NO:57).

117. The IL2 agonist of embodiment 115, wherein the linker comprisestwo, three, four or five repeats of G₄S (SEQ ID NO:57).

118. The IL2 agonist of embodiment 105, which (a) has or comprises anamino acid sequence having the configuration of (e.g., IL2moiety—optional linker—IL2Rα—optional linker—Fc domain) or (b) comprisesthe amino acid sequence of IL2M2.

119. The IL2 agonist of embodiment 105, which (a) has or comprises anamino acid sequence having the configuration of (e.g., IL2amoiety-optional linker—IL2R-optional linker—Fc domain) or (b) comprisesthe amino acid sequence of IL2M3.

120. The IL2 agonist of any one of embodiments 1 to 119, which comprisesa targeting moiety.

121. The IL2 agonist of embodiment 120, wherein the targeting moiety:

-   -   (a) binds to a tumor associated antigen;    -   (b) binds to a tumor microenvironment antigen;    -   (c) binds to a cell surface molecule of tumor reactive        lymphocytes;    -   (d) binds to a checkpoint inhibitor;    -   (e) binds to a peptide-MHC complex;    -   (f) is a peptide-MHC complex; or    -   (g) binds to an antigen associated with or targeted by an        autoimmune response.

122. The IL2 agonist of embodiment 121, wherein the targeting moietybinds to a tumor associated antigen.

123. The IL2 agonist of embodiment 122, wherein the tumor associatedantigen is Fibroblast Activation Protein (FAP), the A1 domain ofTenascin-C (TNC A1), the A2 domain of Tenascin-C (TNC A2), the ExtraDomain B of Fibronectin (EDB), the Melanoma-associated ChondroitinSulfate Proteoglycan (MCSP), MART-1/Melan-A, gp100, Dipeptidyl peptidaseIV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b,colorectal associated antigen (CRC)-C017-1A/GA733, CarcinoembryonicAntigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1Prostate Specific Antigen (PSA) or an immunogenic epitopes thereoPSA-1,PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cellreceptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3),MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05),GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4,GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LACE-1, NAG, GnT-V,MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1,α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn,gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coliprotein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2gangliosides, viral products such as human papilloma virus proteins,Smad family of tumor antigens, Imp-1, P1A, EBV-encoded nuclear antigen(EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40),SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, c-erbB-2, Her2, EGFR, IGF-1R, CD2(T-cell surface antigen), CD3 (heteromultimer associated with the TCR),CD22 (B-cell receptor), CD23 (low binding affinity IgE receptor), CD30(cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumornecrosis factor receptor), IL-6R-(IL6 receptor), CD20, MCSP, PDGFβR(β-platelet-derived growth factor receptor), ErbB2 epithelial celladhesion molecule (EpCAM), EGFR variant III (EGFRvIII), CD19,disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72,glioma-associated antigen, β-human chorionic gonadotropin,alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, MN-CA IX,human telomerase reverse transcriptase, RU1, RU2 (AS), intestinalcarboxyl esterase, mut hsp70-2, M-CSF, prostase, prostase specificantigen (PSA), PAP, LAGA-1a, p53, prostein, PSMA, surviving andtelomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), ELF2M,neutrophil elastase, ephrin B2, insulin growth factor (IGF1)-I, IGF-II,IGFI receptor, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigens, theextra domain A (EDA) or extra domain B (EDB) of fibronectin, or the A1domain of tenascin-C (TnC A1).

124. The IL2 agonist of embodiment 121 or embodiment 122, wherein thetumor associated antigen is a viral antigen.

125. The IL2 agonist of embodiment 124, wherein the viral antigen isEpstein-Barr virus LMP-1, hepatitis C virus E2 glycoprotein, HIV gp160,or HIV gp120, HPV E6, HPV E7, CMV early membrane antigen (EMA) or CMVlate membrane antigen (LMA).

126. The IL2 agonist of embodiment 121, wherein the targeting moietybinds to a tumor microenvironment antigen.

127. The IL2 agonist of embodiment 126, wherein the tumormicroenvironment antigen is an extracellular matrix protein.

128. The IL2 agonist of embodiment 127, wherein the extracellular matrixprotein is syndecan, heparanase, integrins, osteopontin, link,cadherins, laminin, laminin type EGF, lectin, fibronectin, notch,tenascin, collagen and matrixin.

129. The IL2 agonist of embodiment 121, wherein the targeting moietybinds to a cell surface molecule of tumor lymphocytes.

130. The IL2 agonist of embodiment 129, wherein the cell surfacemolecule is CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD1, ICOS,lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, LAG3, TIM3, or B7-H3.

131. The IL2 agonist of embodiment 130, wherein the cell surfacemolecule is PD1.

132. The IL2 agonist of embodiment 131, wherein targeting moiety is ananti-PD1 antibody or antigen binding fragment thereof.

133. The IL2 agonist of embodiment 132, wherein the anti-PD1 antibody orantigen binding fragment thereof inhibits PD1 signaling.

134. The IL2 agonist of embodiment 132, wherein the anti-PD1 antibody orantigen binding fragment thereof does not inhibit PD1 signaling.

135. The IL2 agonist of embodiment 130, wherein the cell surfacemolecule is LAG3.

136. The IL2 agonist of embodiment 121, wherein the targeting moietybinds to a checkpoint inhibitor.

137. The IL2 agonist of embodiment 136, wherein the checkpoint inhibitoris CTLA-4, PD1, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9,LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, VISTA, PSGL1, or CHK2.

138. The IL2 agonist of embodiment 137, wherein the checkpoint inhibitoris PD1.

139. The IL2 agonist of embodiment 138, wherein targeting moiety is ananti-PD1 antibody or antigen binding fragment thereof.

140. The IL2 agonist of embodiment 139, wherein the anti-PD1 antibody orantigen binding fragment thereof inhibits PD1 signaling.

141. The IL2 agonist of embodiment 139, wherein the anti-PD1 antibody orantigen binding fragment thereof does not inhibit PD1 signaling.

142. The IL2 agonist of embodiment 137, wherein the checkpoint inhibitoris LAG3.

143. The IL2 agonist of embodiment 138, wherein the targeting moietybinds to an MHC-peptide complex.

144. The IL2 agonist of embodiment 143 wherein the peptide in thepeptide-MHC complex comprises a tumor neoantigen.

145. The IL2 agonist of embodiment 144, wherein the tumor neoantigen isLCMV derived peptide gp33-41, APF (126-134), BALF (276-284), CEA(571-579), CMV pp65 (495-503), FLU-M1 (58-66), gp100 (154-162), gp100(209-217), HBV Core (18-27), Her2/neu (369-377; V2v9); HPV E7 (11-20),HPV E7 (11-19), HPV E7 (82-90), KLK4 (11-19), LMP1 (125-133), MAG-A3(112-120), NYESO1 (157-165, C165A), NYESO1 (157-165, C165V), p54 WT(264-272), PAP-3 (136-143), PSMA (4-12), PSMA (135-145), Survivin(96-014), Tyrosinase (369-377, 371D), or WT1 (126-134).

146. The IL2 agonist of any one of embodiments 143 to 145, wherein thetargeting moiety comprises an antibody or antigen binding fragmentthereof having complementarity determining regions (“CDRs”) comprising:

-   -   (a) a CDR-H1 having an amino acid sequence selected from any of        SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180,        196, 212, 220, 236, 252, 268, 284, 300, 316, 332, 348, 364, 380,        396, 412, 428, 444, 460, 476, 492, 508, and 524 of International        Patent Publication No. WO 2019005897 A1, which are incorporated        by reference herein;    -   (b) a CDR-H2 having an amino acid sequence selected from any of        SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182,        198, 214, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366, 382,        414, 430, 446, 462, 478, 494, 510, and 526 of International        Patent Publication No. WO 2019005897 A1, which are incorporated        by reference herein;    -   (c) a CDR-H3 having an amino acid sequence selected from any of        SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184,        200, 216, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368, 384,        400, 416, 432, 448, 464, 480, 496, 512, and 528 of International        Patent Publication No. WO 2019005897 A1, which are incorporated        by reference herein;    -   (d) a CDR-L1 having an amino acid sequence selected from any of        SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172,        188, 204, 204, 228, 244, 260, 276, 292, 308, 324, 340, 356, 372,        388, 404, 420, 436, 452, 468, 484, 500, 516, and 532 of        International Patent Publication No. WO 2019005897 A1, which are        incorporated by reference herein;    -   (e) a CDR-L2 having an amino acid sequence selected from any of        SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158,174, 190,        206, 230, 246, 262, 278, 294, 310, 326, 342, 358, 374, 390, 406,        422, 438, 454, 470, 486, 502, 518, and 534 of International        Patent Publication No. WO 2019005897 A1, which are incorporated        by reference herein; and    -   (f) a CDR-L3 having an amino acid sequence selected from an of        SEQ ID NOs: 16. 32, 48, 64, 80, 96, 112, 128, 144,160, 176, 192,        208, 232, 248, 264, 280, 296, 312, 328, 344, 360, 376, 392, 408,        424, 440, 456, 472, 488, 504, 520, and 536 of International        Patent Publication No. WO 2019005897 A1, which are incorporated        by reference herein.

147. The IL2 agonist of embodiment 146, wherein the antibody or antigenbinding fragment has VH-VL amino acid sequences selected from any of SEQID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122,130/138, 146/154, 162/170, 178/186, 194/202, 210/202, 218/226, 234/242,250/258, 266/274, 282/290, 298/306, 314/322, 330/338, 346/354, 362/370,378/386, 394/402, 410/418, 426/434, 442/450, 458/466, 474/482, 490/498,506/514, and 522/530 of International Patent Publication No. WO2019005897 A1, which are incorporated by reference herein.

148. The IL2 agonist of embodiment 147, wherein the antibody or antigenbinding fragment has VH-VL amino acid sequences selected from any of SEQID NOs: 2/10, 34/42, 82/90, 194/202, 282/290, and 506/514 ofInternational Patent Publication No. WO 2019005897 A1, which areincorporated by reference herein.

149. The IL2 agonist of embodiment 121, wherein the targeting moietybinds to an antigen associated with or targeted by an autoimmuneresponse.

150. The IL2 agonist of embodiment 149, wherein the peptide is derivedfrom gliadin, GAD 65, IA-2, insulin B chain, glatiramer acetate (GA),achetylcholine receptor (AChR), p205, insulin, thyroid-stimulatinghormone, tyrosinase, TRP I, or a myelin antigen.

151. The IL2 agonist of embodiment 150, wherein the peptide is derivedfrom IL-4R, IL-6R, or DLL4.

152. The IL2 agonist of any one of embodiments 121 to 151, wherein thetargeting moiety is an antibody or antigen binding fragment thereof.

153. The IL2 agonist of embodiment 152, wherein the targeting moiety isa Fab.

154. The IL2 agonist of embodiment 152, wherein the targeting moiety isa scFv.

155. The IL2 agonist of embodiment 121, wherein the targeting moiety isa peptide-MHC complex.

156. The IL2 agonist of embodiment 155, wherein the peptide-MHC complexbinds to the T cell receptor of tumor lymphocytes.

157. The IL2 agonist of embodiment 155 or embodiment 156, wherein thepeptide in the peptide-MHC complex comprises a tumor neoantigen.

158. The IL2 agonist of embodiment 157, wherein the tumor neoantigen isLCMV derived peptide gp33-41, APF (126-134), BALF (276-284), CEA(571-579), CMV pp65 (495-503), FLU-M1 (58-66), gp100 (154-162), gp100(209-217), HBV Core (18-27), Her2/neu (369-377; V2v9); HPV E7 (11-20),HPV E7 (11-19), HPV E7 (82-90), KLK4 (11-19), LMP1 (125-133), MAG-A3(112-120), NYESO1 (157-165, C165A), NYESO1 (157-165, C165V), p54 WT(264-272), PAP-3 (136-143), PSMA (4-12), PSMA (135-145), Survivin(96-014), Tyrosinase (369-377, 371D), or WT1 (126-134).

159. The IL2 agonist of embodiment 155, wherein the peptide inpeptide-MHC complex comprises a viral antigen.

160. The IL2 agonist of embodiment 159, wherein the viral antigen isCMVpp65 or HPV16E7.

161. The IL2 agonist of any one of embodiments 155 to 160, wherein thepeptide-MHC complex further comprises β2 microglobulin or a fragmentthereof.

162. The IL2 agonist of embodiment 161, wherein the peptide MHC complexcomprises a type I MHC domain.

163. The IL2 agonist of embodiment 162, wherein the peptide MHC complexcomprises, in an N- to C-terminal orientation a MHC peptide, a linker, aβ2-microglobulin domain, a linker, and a type I MHC domain.

164. The IL2 agonist of embodiment 163, wherein the linker connectingthe MHC peptide and the β2-microglobulin domain comprises the amino acidsequence GCGGS (SEQ ID NO:77).

165. The IL2 agonist of any one of embodiment 155 to 160, wherein thepeptide-MHC complex does not comprise β2 microglobulin or a fragmentthereof.

166. The IL2 agonist of embodiment 165, wherein the peptide MHC complexcomprises a type II MHC domain.

167. A nucleic acid or plurality of nucleic acids encoding the IL2agonist of any one of embodiments 1 to 166.

168. A host cell engineered to express the IL2 agonist of any one ofembodiments 1 to 166 or the nucleic acid(s) of embodiment 167.

169. A method of producing the IL2 agonist of any one of embodiments 1to 166, comprising culturing the host cell of embodiment 168 andrecovering the IL2 agonist expressed thereby.

170. A pharmaceutical composition comprising the IL2 agonist of any oneof embodiments 1 to 166 and an excipient.

171. A method of treating cancer, comprising administering to a subjectin need thereof the IL2 agonist of any one of embodiments 1 to 166 orthe pharmaceutical composition of embodiment 170.

172. A method of treating cancer, comprising administering to a subjectin need thereof:

-   -   (a) chimeric antigen receptor (“CAR”) T cells (“CART cells”);        and    -   (b) an IL2 agonist comprising a targeting moiety that binds to        the T cell receptor of the CART cells or another cell surface        molecule on the CART cells, optionally wherein the targeting        moiety is capable of binding to extracellular domain of the CAR.

173. The method of embodiment 172, wherein the IL2 agonist is monovalentfor an IL2 moiety and/or the targeting moiety.

174. The method of embodiment 172 or embodiment 173, wherein the IL2agonist is an IL2 agonist according to any one of embodiments 1 to 166,preferably an IL2 agonist according to any one of embodiments 94 to 97,121, and 155 to 166.

175. The method of any one of embodiments 172 to 174, wherein the CARTcells are not engineered to express a variant IL2-Rβ receptor.

176. The method of any one of embodiments 172 to 174, wherein the CARTcells are not engineered to express any variant IL2 receptor.

177. The method of any one of embodiments 172 to 176, wherein the IL2agonist is administered to the subject within one week of administrationof the CART cells.

178. The method of embodiment 177, wherein the wherein the IL2 agonistis administered to the subject on the same day as the administration ofthe CART cells.

179. The method of any one of embodiments 172 to 178, which comprisesdosing the subject with the IL2 agonist for a period of at least twoweeks.

180. The method of embodiment 179, wherein the IL2 agonist is dosed bycontinuous infusion.

181. The method of embodiment 179, wherein the IL2 agonist is dosed bydaily administration for at least a portion of the at least two-weekperiod.

182. The method of embodiment 179, wherein the IL2 agonist is dosedaccording to a split dosing regimen, comprising:

-   -   (a) administering the IL2 agonist at a first dosing frequency in        the initial part of the at least two week period; and    -   (b) administering IL2 agonist at a second dosing frequency in a        subsequent portion of the at least two week period.

183. The method of embodiment 182, wherein the first dosing frequency isdaily.

184. The method of embodiment 182 or embodiment 183, wherein the seconddosing frequency is less frequent than the first dosing frequency.

185. The method of embodiment 184, wherein the second dosing frequencyis weekly.

186. The method of any one of embodiments 182 to 185, wherein thesubject is transitioned from the first dosing frequency to the seconddosing frequency concurrently with or after exhaustion of the CARTcells.

187. The method of any one of embodiments 171 to 186, which furthercomprises administering an anti-PD1 antibody to the subject.

188. The method of embodiment 187, wherein the anti-PD1 antibody isMDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514),PDR001, REGN2810, or BGB-108.

189. The method of any one of embodiments 172 to 188, wherein the CAR isdesigned to target any of the targets identified in Section 6.11.1.3.

190. The method of any one of embodiments 172 to 189, wherein the CAR isconfigured according to Section 6.11.1.1 and subsections thereof.

191. The method of any one of embodiments 172 to 190, wherein thetargeting moiety comprises a pMHC recognized by the antigen bindingdomain of the CAR.

192. A method of treating autoimmune disease, comprising administeringto a subject in need thereof:

-   -   (a) chimeric antigen receptor (“CAR”) T cells (“CART cells”);        and    -   (b) an IL2 agonist comprising a targeting moiety that binds to a        cell surface antigen present on the CART cells, optionally        wherein the targeting moiety is capable of binding to        extracellular domain of the CAR.

193. The method of embodiment 192, wherein the IL2 agonist is monovalentfor an IL2 moiety and/or the targeting moiety.

194. The method of embodiment 192 or embodiment 193, wherein the IL2agonist is an IL2 agonist according to any one of embodiments 1 to 166,preferably an IL2 agonist according to any one of embodiments 94 to 97,121, and 155 to 166 according to any one of embodiments 94 to 97, 121,and 155 to 166.

195. The method of any one of embodiments 192 to 194, wherein thetargeting moiety comprises a pMHC cloned from an autoimmune target celland/or which is recognized by the antigen binding domain of the CAR.

196. The method of any one of embodiments 192 to 195, wherein the CARTcell is a Treg cell.

197. The method of any one of embodiments 192 to 196, wherein the CAR isdesigned to target any of the targets identified in Section 6.11.1.4.

198. The method of any one of embodiments 192 to 197, wherein the CAR isconfigured according to Section 6.11.1.1 and subsections thereof.

199. An IL2 agonist comprising:

-   -   (a) an Fc domain; an    -   (b) an IL2 moiety C-terminal to the Fc domain, wherein the IL2        moiety comprises an IL2 domain and an IL2-Rα domain,

optionally wherein the IL2 agonist has one or more features of the IL2agonists of any one of embodiments 1 to 36.

200. The IL2 agonist of embodiment 199, wherein the IL2 domain isN-terminal to the IL2-Rα domain.

201. The IL2 agonist of embodiment 199, wherein the IL2 domain isC-terminal to the IL2-Rα domain.

202. The IL2 agonist of any one of embodiments 199 to 201, wherein theIL2 moiety comprises an IL2 domain comprising an amino acid sequencehaving:

-   -   (a) at least about 90% or at least about 95% sequence identity        to mature human IL2,    -   (b) an N-terminal alanine deletion as compared to mature human        IL2;    -   (c) an IL2 variant which has an amino acid substitution at        position N88 as compared to wild type IL2, optionally wherein        the amino acid substitution is N88D;    -   (d) the amino acid substitution C125S, C125A or C125V as        compared to wild type IL2; or    -   (e) any combination of (a), (b), (c) and/or (d).

203. The IL2 agonist of any one of embodiments 199 to 202, wherein theIL2-Rα domain comprises or consists of an amino acid sequence having atleast about 90%, at least about 95%, at least about 96%, at least about97%, at least about 98%, at least about 99% or 100% sequence identity toan IL2 binding portion of human IL-2Rα, optionally wherein said bindingportion has:

-   -   (a) at least 160 amino acids, at least 161 amino acids, at least        162 amino acids, at least 164 amino acids or at least 165 amino        acids of human IL2-Rα; and/or    -   (b) up to 251, up to 240, up to 230, up to 220, up to 210, up to        200, up to 190, up to 180 or up to 170 amino acids of the        extracellular domain of human IL2-Rα.

204. The IL2 agonist of any one of embodiments 199 to 203, wherein theIL2 domain and the IL2-Rα domain are connected via a linker (“the IL2moiety linker”).

205. The IL2 agonist of embodiment 204, wherein the IL2 moiety linker isat least 10 or at least 15 amino acids in length.

206. The IL2 agonist of embodiment 204 or embodiment 205, wherein theIL2 moiety linker is or comprises a glycine-serine linker.

207. The IL2 agonist of any one of embodiments 204 to 206, wherein theIL2 moiety linker comprises the amino acid sequence G₄S (SEQ ID NO:57).

208. The IL2 agonist of embodiment 207, wherein the IL2 moiety linker isor comprises a multimer of the amino acid sequence G₄S (SEQ ID NO:57).

209. The IL2 agonist of embodiment 208, wherein the multimer comprises2, 3, 4, 5, 6 or more repeats of the amino acid sequence G₄S (SEQ IDNO:57).

210. The IL2 agonist of any one of embodiments 199 to 209, wherein theFc domain and the IL2 moiety are connected via a linker (the “Fc-IL2linker”).

211. The IL2 agonist of embodiment 210, wherein the Fc-IL2 linker is atleast 5 or at least 10 amino acids in length.

212. The IL2 agonist of embodiment 210 or embodiment 211, wherein theFc-IL2 linker is or comprises a glycine-serine linker.

213. The IL2 agonist of any one of embodiments 210 to 212, wherein theFc-IL2 linker comprises the amino acid sequence G₄S (SEQ ID NO:57).

214. The IL2 agonist of embodiment 207, wherein the Fc-IL2 linker is orcomprises a multimer of the amino acid sequence G₄S (SEQ ID NO:57).

215. The IL2 agonist of embodiment 208, wherein the multimer comprises2, 3, 4, 5, 6 or more repeats of the amino acid sequence G₄S (SEQ IDNO:57).

216. The IL2 agonist of any one of embodiments 199 to 215, wherein theFc domain is an IgG1, IgG2, IgG3 or IgG4 Fc domain.

217. The IL2 agonist of embodiment 216, wherein the Fc domain hasreduced effector function.

218. The IL2 agonist of any one of embodiments 199 to 217 which is adimer. 219. The IL2 agonist of embodiment 218, which is a homodimer.

220. The IL2 agonist of embodiment 218, which is a heterodimer.

221. The IL2 agonist of any one of embodiments 199 to 220, which isbivalent for the IL2 moiety.

222. The IL2 agonist of any one of embodiments 199 to 221, whichcomprises a targeting moiety.

223. The IL2 agonist of embodiment 222, wherein the targeting moiety isN-terminal to the Fc domain.

224. The IL2 agonist of embodiment 222 or embodiment 223, wherein thetargeting moiety:

-   -   (a) binds to a tumor associated antigen;    -   (b) binds to a tumor microenvironment antigen;    -   (c) binds to a cell surface molecule of tumor reactive        lymphocytes;    -   (d) binds to a checkpoint inhibitor;    -   (e) binds to a peptide-MHC complex;    -   (f) is a peptide-MHC complex; or    -   (g) binds to an antigen associated with or targeted by an        autoimmune response.

225. The IL2 agonist of embodiment 224, wherein the targeting moietybinds to a tumor associated antigen.

226. The IL2 agonist of embodiment 225, wherein the tumor associatedantigen is Fibroblast Activation Protein (FAP), the A1 domain ofTenascin-C (TNC A1), the A2 domain of Tenascin-C (TNC A2), the ExtraDomain B of Fibronectin (EDB), the Melanoma-associated ChondroitinSulfate Proteoglycan (MCSP), MART-1/Melan-A, gp100, Dipeptidyl peptidaseIV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b,colorectal associated antigen (CRC)-C017-1A/GA733, CarcinoembryonicAntigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1Prostate Specific Antigen (PSA) or an immunogenic epitopes thereoPSA-1,PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cellreceptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3),MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05),GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4,GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V,MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1,α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn,gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coliprotein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2gangliosides, viral products such as human papilloma virus proteins,Smad family of tumor antigens, Imp-1, PIA, EBV-encoded nuclear antigen(EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40),SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, c-erbB-2, Her2, EGFR, IGF-1R, CD2(T-cell surface antigen), CD3 (heteromultimer associated with the TCR),CD22 (B-cell receptor), CD23 (low binding affinity IgE receptor), CD30(cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumornecrosis factor receptor), IL-6R-(IL6 receptor), CD20, MCSP, PDGFβR(β-platelet-derived growth factor receptor), ErbB2 epithelial celladhesion molecule (EpCAM), EGFR variant III (EGFRvIII), CD19,disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72,glioma-associated antigen, β-human chorionic gonadotropin,alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, MN-CA IX,human telomerase reverse transcriptase, RU1, RU2 (AS), intestinalcarboxyl esterase, mut hsp70-2, M-CSF, prostase, prostase specificantigen (PSA), PAP, LAGA-1a, p53, prostein, PSMA, surviving andtelomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), ELF2M,neutrophil elastase, ephrin B2, insulin growth factor (IGF1)-I, IGF-II,IGFI receptor, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigens, theextra domain A (EDA) or extra domain B (EDB) of fibronectin, or the A1domain of tenascin-C (TnC A1).

227. The IL2 agonist of embodiment 224 or embodiment 225, wherein thetumor associated antigen is a viral antigen.

228. The IL2 agonist of embodiment 227, wherein the viral antigen isEpstein-Barr virus LMP-1, hepatitis C virus E2 glycoprotein, HIV gp160,or HIV gp120, HPV E6, HPV E7, CMV early membrane antigen (EMA) or CMVlate membrane antigen (LMA).

229. The IL2 agonist of embodiment 224, wherein the targeting moietybinds to a tumor microenvironment antigen.

230. The IL2 agonist of embodiment 229, wherein the tumormicroenvironment antigen is an extracellular matrix protein.

231. The IL2 agonist of embodiment 230, wherein the extracellular matrixprotein is syndecan, heparanase, integrins, osteopontin, link,cadherins, laminin, laminin type EGF, lectin, fibronectin, notch,tenascin, collagen and matrixin.

232. The IL2 agonist of embodiment 224, wherein the targeting moietybinds to a cell surface molecule of tumor lymphocytes.

233. The IL2 agonist of embodiment 232, wherein the cell surfacemolecule is CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD1, ICOS,lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, LAG3, TIM3, or B7-H3.

234. The IL2 agonist of embodiment 233, wherein the cell surfacemolecule is PD1.

235. The IL2 agonist of embodiment 234, wherein targeting moiety is ananti-PD1 antibody or antigen binding fragment thereof.

236. The IL2 agonist of embodiment 233, wherein the cell surfacemolecule is LAG3.

237. The IL2 agonist of embodiment 224, wherein the targeting moietybinds to a checkpoint inhibitor.

238. The IL2 agonist of embodiment 237, wherein the checkpoint inhibitoris CTLA-4, PD1, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9,LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, VISTA, PSGL1, or CHK2.

239. The IL2 agonist of embodiment 238, wherein the checkpoint inhibitoris PD1.

240. The IL2 agonist of embodiment 238, wherein the checkpoint inhibitoris LAG3.

241. The IL2 agonist of embodiment 239, wherein the targeting moietybinds to an MHC-peptide complex.

242. The IL2 agonist of embodiment 241 wherein the peptide in thepeptide-MHC complex comprises a tumor neoantigen.

243. The IL2 agonist of embodiment 242, wherein the tumor neoantigen isLCMV derived peptide gp33-41, APF (126-134), BALF (276-284), CEA(571-579), CMV pp65 (495-503), FLU-M1 (58-66), gp100 (154-162), gp100(209-217), HBV Core (18-27), Her2/neu (369-377; V2v9); HPV E7 (11-20),HPV E7 (11-19), HPV E7 (82-90), KLK4 (11-19), LMP1 (125-133), MAG-A3(112-120), NYESO1 (157-165, C165A), NYESO1 (157-165, C165V), p54 WT(264-272), PAP-3 (136-143), PSMA (4-12), PSMA (135-145), Survivin(96-014), Tyrosinase (369-377, 371D), or WT1 (126-134).

244. The IL2 agonist of any one of embodiments 224 to 243, wherein thetargeting moiety is an antibody or antigen binding fragment thereof.

245. The IL2 agonist of embodiment 244, wherein the targeting moiety isa Fab. 246. The IL2 agonist of embodiment 244, wherein the targetingmoiety is a scFv. 247. The IL2 agonist of embodiment 224, wherein thetargeting moiety is a peptide-MHC complex.

248. The IL2 agonist of embodiment 247, wherein the peptide-MHC complexbinds to the T cell receptor of tumor lymphocytes.

249. The IL2 agonist of embodiment 247 or embodiment 248, wherein thepeptide in the peptide-MHC complex comprises a tumor neoantigen.

250. The IL2 agonist of embodiment 249, wherein the tumor neoantigen isLCMV derived peptide gp33-41, APF (126-134), BALF (276-284), CEA(571-579), CMV pp65 (495-503), FLU-M1 (58-66), gp100 (154-162), gp100(209-217), HBV Core (18-27), Her2/neu (369-377; V2v9); HPV E7 (11-20),HPV E7 (11-19), HPV E7 (82-90), KLK4 (11-19), LMP1 (125-133), MAG-A3(112-120), NYESO1 (157-165, C165A), NYESO1 (157-165, C165V), p54 WT(264-272), PAP-3 (136-143), PSMA (4-12), PSMA (135-145), Survivin(96-014), Tyrosinase (369-377, 371D), or WT1 (126-134).

251. The IL2 agonist of embodiment 247, wherein the peptide inpeptide-MHC complex comprises a viral antigen.

252. The IL2 agonist of embodiment 251, wherein the viral antigen isCMVpp65 or HPV16E7.

253. The IL2 agonist of any one of embodiments 247 to 252, wherein thepeptide-MHC complex further comprises β2 microglobulin or a fragmentthereof.

254. The IL2 agonist of embodiment 253, wherein the peptide MHC complexcomprises a type I MHC domain.

255. The IL2 agonist of embodiment 254, wherein the peptide MHC complexcomprises, in an N- to C-terminal orientation a MHC peptide, a linker, aβ2-microglobulin domain, a linker, and a type I MHC domain.

256. The IL2 agonist of embodiment 255, wherein the linker connectingthe MHC peptide and the β2-microglobulin domain comprises the amino acidsequence GCGGS.

257. The IL2 agonist of any one of embodiment 247 to 252, wherein thepeptide-MHC complex does not comprise β2 microglobulin or a fragmentthereof.

258. The IL2 agonist of embodiment 257, wherein the peptide MHC complexcomprises a type II MHC domain.

259. A nucleic acid or plurality of nucleic acids encoding the IL2agonist of any one of embodiments 199 to 258.

260. A host cell engineered to express the IL2 agonist of any one ofembodiments 199 to 258 or the nucleic acid(s) of embodiment 259.

261. A method of producing the IL2 agonist of any one of embodiments 199to 258, comprising culturing the host cell of embodiment 260 andrecovering the IL2 agonist expressed thereby.

262. A pharmaceutical composition comprising the IL2 agonist of any oneof embodiments 199 to 258 and an excipient.

263. A method of treating cancer, comprising administering to a subjectin need thereof the IL2 agonist of any one of embodiments 199 to 258 orthe pharmaceutical composition of embodiment 262.

264. A method of treating cancer, comprising administering to a subjectin need thereof:

-   -   (a) chimeric antigen receptor (“CAR”) T cells (“CART cells”);        and    -   (b) an IL2 agonist according to any one of embodiments 199 to        224 comprising a targeting moiety that binds to the T cell        receptor of the CART cells or another cell surface molecule on        the CART cells, optionally wherein the targeting moiety is        capable of binding to the extracellular domain of the CAR,

optionally wherein the CAR:

-   -   (i) wherein the CAR is designed to target any of the targets        identified in Section 6.11.1.4; and/or    -   (ii) wherein the CAR is configured according to Section 6.11.1.1        and subsections thereof.

265. The method of embodiment 263 or embodiment 264, which furthercomprises administering an anti-PD1 antibody to the subject.

266. The method of embodiment 265, wherein the anti-PD1 antibody isMDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514),PDR001, REGN2810, or BGB-108.

267. An IL2 agonist comprising:

-   -   (a) a first polypeptide chain comprising:        -   (i) a targeting moiety, e.g., a targeting moiety capable of            binding to a cell surface molecule on a chimeric antigen            receptor (CAR) T cell, optionally wherein the targeting            moiety is capable of binding to the extracellular domain of            the CAR; and        -   (ii) a first Fc domain C-terminal to the targeting moiety;            and    -   (b) a second polypeptide chain comprising:        -   (i) an IL2 moiety; and        -   (ii) a second Fc domain N- or C-terminal to the IL2 moiety,

optionally wherein the IL2 agonist has one or more features of the IL2agonists of any one of embodiments 1 to 36.

268. The IL2 agonist of embodiment 267, which is a dimer.

269. The IL2 agonist of embodiment 268, which is a homodimer.

270. The IL2 agonist of embodiment 269, which is bivalent for thetargeting moiety and the IL2 moiety.

271. The IL2 agonist of embodiment 270, which has the configurationdepicted in FIG. 22 .A.1.

272. The IL2 agonist of embodiment 270 or embodiment 271, whichcomprises:

-   -   (a) a first polypeptide chain comprising:        -   (i) a first targeting moiety capable of binding to a            chimeric antigen receptor;        -   (ii) an optional linker;        -   (iii) a first Fc domain C-terminal to the targeting moiety;        -   (iv) an optional linker; and        -   (v) a first IL2 moiety C-terminal to the first Fc domain;    -   (b) a second polypeptide chain comprising:        -   (i) a second targeting moiety capable of binding to a            chimeric antigen receptor;        -   (ii) an optional linker;        -   (iii) a second Fc domain C-terminal to the targeting moiety;        -   (iv) an optional linker; and        -   (v) a second IL2 moiety C-terminal to the first Fc domain.

273. The IL2 agonist of embodiment 268, which is a heterodimer.

274. The IL2 agonist of embodiment 273, which is monovalent for thetargeting moiety and the IL2 moiety.

275. The IL2 agonist of embodiment 274, which has the configurationdepicted in FIG. 22 .A.2.

276. The IL2 agonist of embodiment 274 or embodiment 274, whichcomprises:

-   -   (a) a first polypeptide chain comprising:        -   (i) a targeting moiety capable of binding to a chimeric            antigen receptor;        -   (ii) an optional linker; and        -   (iii) a first Fc domain C-terminal to the targeting moiety;            and    -   (b) a second polypeptide chain comprising:        -   (i) an IL2 moiety;        -   (ii) an optional linker; and        -   (iii) a second Fc domain C-terminal to the IL2 moiety.

277. The IL2 agonist of any one of embodiments 267 to 276, wherein theIL2 moiety and/or the IL2 agonist has attenuated binding to humanIL2-Rβ.

278. The IL2 agonist of any one of embodiments 267 to 277, wherein theIL2 moiety and/or the IL2 agonist has attenuated binding to humanIL2-Rα.

279. The IL2 agonist of any one of embodiments 267 to 278, wherein theIL2 moiety and/or the IL2 agonist has:

-   -   (a) 50-fold to 1000-fold attenuated binding to human IL2-Rβ as        compared to wild type IL2; and/or    -   (b) up to 100-fold attenuated binding to human IL2-Rα as        compared to wild-type human IL2.

280. The IL2 agonist of any one of embodiments 267 to 279, wherein theIL2 moiety and/or the IL2 agonist has attenuated binding affinity to thehigh affinity IL2 receptor compared to wild type IL2.

281. The IL2 agonist of embodiment 280, wherein the binding affinity isattenuated by 10-fold to 1,000-fold.

282. The IL2 agonist of any one of embodiments 267 to 281, wherein theIL2 moiety comprises an IL2 domain comprising an amino acid sequencehaving:

-   -   (a) at least about 90% or at least about 95% sequence identity        to mature human IL2,    -   (b) an N-terminal alanine deletion as compared to mature human        IL2;    -   (c) the amino acid substitution C125S, C125A or C125V as        compared to wild type IL2;    -   (d) the amino acid substitutions H16A and/or F42A as compared to        wild type IL2; or    -   (e) any combination of (a), (b), (c) and/or (d).

283. The IL2 agonist of any one of embodiments 267 to 282, which lacksan IL2 binding portion of IL2-Rα.

284. The IL2 agonist of any one of embodiments 267 to 283, wherein theFc domain is an IgG1, IgG2, IgG3 or IgG4 Fc domain.

285. The IL2 agonist of embodiment 284, wherein the Fc domain hasreduced effector function.

286. The IL2 agonist of any one of embodiments 1 to 285, wherein thetargeting moiety is a peptide-MHC complex.

287. The IL2 agonist of embodiment 286, wherein the peptide-MHC complexbinds to the T cell receptor of tumor lymphocytes.

288. The IL2 agonist of embodiment 286 or embodiment 287, wherein thepeptide in the peptide-MHC complex comprises a tumor neoantigen.

289. The IL2 agonist of embodiment 288, wherein the tumor neoantigen isLCMV derived peptide gp33-41, APF (126-134), BALF (276-284), CEA(571-579), CMV pp65 (495-503), FLU-M1 (58-66), gp100 (154-162), gp100(209-217), HBV Core (18-27), Her2/neu (369-377; V2v9); HPV E7 (11-20),HPV E7 (11-19), HPV E7 (82-90), KLK4 (11-19), LMP1 (125-133), MAG-A3(112-120), NYESO1 (157-165, C165A), NYESO1 (157-165, C165V), p54 WT(264-272), PAP-3 (136-143), PSMA (4-12), PSMA (135-145), Survivin(96-014), Tyrosinase (369-377, 371D), or WT1 (126-134).

290. The IL2 agonist of embodiment 286 or embodiment 287, wherein thepeptide in peptide-MHC complex comprises a viral antigen.

291. The IL2 agonist of embodiment 290, wherein the viral antigen isCMVpp65 or HPV16E7.

292. The IL2 agonist of any one of embodiments 286 to 291, wherein thepeptide-MHC complex further comprises β2 microglobulin or a fragmentthereof.

293. The IL2 agonist of embodiment 292, wherein the peptide MHC complexcomprises a type I MHC domain.

294. The IL2 agonist of embodiment 293, wherein the peptide MHC complexcomprises, in an N- to C-terminal orientation a MHC peptide, a linker, aβ2-microglobulin domain, a linker, and a type I MHC domain.

295. The IL2 agonist of embodiment 294, wherein the linker connectingthe MHC peptide and the β2-microglobulin domain comprises the amino acidsequence GCGGS (SEQ ID NO:77).

296. The IL2 agonist of any one of embodiment 286 to 291, wherein thepeptide-MHC complex does not comprise β2 microglobulin or a fragmentthereof.

297. The IL2 agonist of embodiment 296, wherein the peptide MHC complexcomprises a type II MHC domain.

298. The IL2 agonist of any one of embodiments 267 to 285, wherein thetargeting moiety is an antibody or antigen binding fragment thereof.

299. The IL2 agonist of embodiment 298, wherein the targeting moiety isa Fab and wherein the IL2 agonist comprises a third polypeptide chaincomprising the light chain of the Fab.

300. The IL2 agonist of embodiment 298, wherein the targeting moiety isa scFv.

301. The IL2 agonist of any one of embodiments 267 to 285 and 298 to300, wherein the targeting moiety:

-   -   (a) binds to a tumor associated antigen;    -   (b) binds to a tumor microenvironment antigen;    -   (c) binds to a cell surface molecule of tumor reactive        lymphocytes;    -   (d) binds to a checkpoint inhibitor;    -   (e) binds to a peptide-MHC complex;    -   (f) binds to an antigen associated with or targeted by an        autoimmune response.

302. The IL2 agonist of embodiment 301, wherein the targeting moietybinds to a tumor associated antigen.

303. The IL2 agonist of embodiment 302, wherein the tumor associatedantigen is Fibroblast Activation Protein (FAP), the A1 domain ofTenascin-C (TNC A1), the A2 domain of Tenascin-C (TNC A2), the ExtraDomain B of Fibronectin (EDB), the Melanoma-associated ChondroitinSulfate Proteoglycan (MCSP), MART-1/Melan-A, gp100, Dipeptidyl peptidaseIV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b,colorectal associated antigen (CRC)-C017-1A/GA733, CarcinoembryonicAntigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1Prostate Specific Antigen (PSA) or an immunogenic epitopes thereoPSA-1,PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cellreceptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3),MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05),GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4,GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LACE-1, NAG, GnT-V,MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1,α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn,gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coliprotein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2gangliosides, viral products such as human papilloma virus proteins,Smad family of tumor antigens, Imp-1, P1A, EBV-encoded nuclear antigen(EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40),SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, c-erbB-2, Her2, EGFR, IGF-1R, CD2(T-cell surface antigen), CD3 (heteromultimer associated with the TCR),CD22 (B-cell receptor), CD23 (low binding affinity IgE receptor), CD30(cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumornecrosis factor receptor), IL-6R-(IL6 receptor), CD20, MCSP, PDGFβR(β-platelet-derived growth factor receptor), ErbB2 epithelial celladhesion molecule (EpCAM), EGFR variant III (EGFRvIII), CD19,disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72,glioma-associated antigen, β-human chorionic gonadotropin,alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, MN-CA IX,human telomerase reverse transcriptase, RU1, RU2 (AS), intestinalcarboxyl esterase, mut hsp70-2, M-CSF, prostase, prostase specificantigen (PSA), PAP, LAGA-1a, p53, prostein, PSMA, surviving andtelomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), ELF2M,neutrophil elastase, ephrin B2, insulin growth factor (IGF1)-I, IGF-II,IGFI receptor, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigens, theextra domain A (EDA) or extra domain B (EDB) of fibronectin, or the A1domain of tenascin-C (TnC A1).

304. The IL2 agonist of embodiment 301 or embodiment 302, wherein thetumor associated antigen is a viral antigen.

305. The IL2 agonist of embodiment 304, wherein the viral antigen isEpstein-Barr virus LMP-1, hepatitis C virus E2 glycoprotein, HIV gp160,or HIV gp120, HPV E6, HPV E7, CMV early membrane antigen (EMA) or CMVlate membrane antigen (LMA).

306. The IL2 agonist of embodiment 301, wherein the targeting moietybinds to a tumor microenvironment antigen.

307. The IL2 agonist of embodiment 306, wherein the tumormicroenvironment antigen is an extracellular matrix protein.

308. The IL2 agonist of embodiment 307, wherein the extracellular matrixprotein is syndecan, heparanase, integrins, osteopontin, link,cadherins, laminin, laminin type EGF, lectin, fibronectin, notch,tenascin, collagen and matrixin.

309. The IL2 agonist of embodiment 301, wherein the targeting moietybinds to a cell surface molecule of tumor lymphocytes.

310. The IL2 agonist of embodiment 309, wherein the cell surfacemolecule is CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD1, ICOS,lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, LAG3, TIM3, or B7-H3.

311. The IL2 agonist of embodiment 310, wherein the cell surfacemolecule is PD1.

312. The IL2 agonist of embodiment 310, wherein the cell surfacemolecule is LAG3.

313. The IL2 agonist of embodiment 301, wherein the targeting moietybinds to a checkpoint inhibitor.

314. The IL2 agonist of embodiment 313, wherein the checkpoint inhibitoris CTLA-4, PD1, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9,LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, VISTA, PSGL1, or CHK2.

315. The IL2 agonist of embodiment 314, wherein the checkpoint inhibitoris PD1.

316. The IL2 agonist of embodiment 314, wherein the checkpoint inhibitoris LAG3.

317. The IL2 agonist of embodiment 301, wherein the targeting moietybinds to an MHC-peptide complex.

318. The IL2 agonist of embodiment 317 wherein the peptide in thepeptide-MHC complex comprises a tumor neoantigen.

319. The IL2 agonist of embodiment 318, wherein the tumor neoantigen isLCMV derived peptide gp33-41, APF (126-134), BALF (276-284), CEA(571-579), CMV pp65 (495-503), FLU-M1 (58-66), gp100 (154-162), gp100(209-217), HBV Core (18-27), Her2/neu (369-377; V2v9); HPV E7 (11-20),HPV E7 (11-19), HPV E7 (82-90), KLK4 (11-19), LMP1 (125-133), MAG-A3(112-120), NYESO1 (157-165, C165A), NYESO1 (157-165, C165V), p54 WT(264-272), PAP-3 (136-143), PSMA (4-12), PSMA (135-145), Survivin(96-014), Tyrosinase (369-377, 371D), or WT1 (126-134).

320. The IL2 agonist of embodiment 301, wherein the targeting moietybinds to an antigen associated with or targeted by an autoimmuneresponse.

321. The IL2 agonist of embodiment 320, wherein the peptide is derivedfrom gliadin, GAD 65, IA-2, insulin B chain, glatiramer acetate (GA),achetylcholine receptor (AChR), p205, insulin, thyroid-stimulatinghormone, tyrosinase, TRP I, or a myelin antigen.

322. The IL2 agonist of embodiment 321, wherein the peptide is derivedfrom IL-4R, IL-6R, or DLL4.

323. A nucleic acid or plurality of nucleic acids encoding the IL2agonist of any one of embodiments 267 to 322.

324. A host cell engineered to express the IL2 agonist of any one ofembodiments 267 to 322 or the nucleic acid(s) of embodiment 323.

325. A method of producing the IL2 agonist of any one of embodiments 267to 322, comprising culturing the host cell of embodiment 324 andrecovering the IL2 agonist expressed thereby.

326. A pharmaceutical composition comprising the IL2 agonist of any oneof embodiments 267 to 322 and an excipient.

327. A method of treating cancer, comprising administering to a subjectin need thereof the IL2 agonist of any one of embodiments 267 to 322 orthe pharmaceutical composition of embodiment 326.

328. A method of treating cancer, comprising administering to a subjectin need thereof:

-   -   (a) chimeric antigen receptor (“CAR”) T cells (“CART cells”);        and    -   (b) an IL2 agonist according to any one of embodiments 267 to        322 whose targeting moiety binds to a cell surface molecule on        the chimeric antigen receptor (CAR) T cells, optionally wherein        the targeting moiety is capable of binding to the extracellular        domain of the CAR,

optionally wherein the CAR is:

-   -   (i) designed to target any of the targets identified in Section        6.11.1.4; and/or    -   (ii) configured according to Section 6.11.1.1 and subsections        thereof.

329. The method of embodiment 328, wherein the targeting moietycomprises a pMHC recognized by the antigen binding domain of the CAR.

330. The method of embodiment 328 or embodiment 329, wherein the CARTcells are not engineered to express a variant IL2-Rβ receptor.

331. The method of any one of embodiments 328 to 330, wherein the CARTcells are not engineered to express any variant IL2 receptor.

332. The method of any one of embodiments 328 to 331, wherein the IL2agonist is administered to the subject within one week of administrationof the CART cells.

333. The method of embodiment 332, wherein the wherein the IL2 agonistis administered to the subject on the same day as the administration ofthe CART cells.

334. The method of any one of embodiments 328 to 333, which comprisesdosing the subject with the IL2 agonist for a period of at least twoweeks.

335. The method of embodiment 334, wherein the IL2 agonist is dosed bycontinuous infusion.

336. The method of embodiment 334, wherein the IL2 agonist is dosed bydaily administration for at least a portion of the at least two-weekperiod.

337. The method of embodiment 334, wherein the IL2 agonist is dosedaccording to a split dosing regimen, comprising:

-   -   (a) administering the IL2 agonist at a first dosing frequency in        the initial part of the at least two week period; and    -   (b) administering IL2 agonist at a second dosing frequency in a        subsequent portion of the at least two week period.

338. The method of embodiment 337, wherein the first dosing frequency isdaily.

339. The method of embodiment 337 or embodiment 338, wherein the seconddosing frequency is less frequent than the first dosing frequency.

340. The method of embodiment 339, wherein the second dosing frequencyis weekly.

341. The method of any one of embodiments 337 to 340, wherein thesubject is transitioned from the first dosing frequency to the seconddosing frequency concurrently with or after exhaustion of the CARTcells.

342. The method of any one of embodiments 328 to 341, which furthercomprises administering an anti-PD1 antibody to the subject.

343. The method of embodiment 342, wherein the anti-PD1 antibody isMDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514),PDR001, REGN2810, or BGB-108.

344. A method of treating autoimmune disease, comprising administeringto a subject in need thereof:

-   -   (a) chimeric antigen receptor (“CAR”) T cells (“CART cells”);        and    -   (b) an IL2 agonist according to any one of embodiments 267 to        322 whose targeting moiety binds to a cell surface molecule on        the chimeric antigen receptor (CAR) T cells, optionally wherein        the targeting moiety is capable of binding to the extracellular        domain of the CAR,

optionally wherein the CAR is:

-   -   (i) designed to target any of the targets identified in Section        6.11.1.4; and/or    -   (ii) configured according to Section 6.11.1.1 and subsections        thereof.

345. The method of embodiment 344, wherein the targeting moietycomprises a pMHC cloned from an autoimmune target cell.

346. The method of embodiment 344 or embodiment 345, wherein the CARTcell is a Treg cell.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.In the event that there is an inconsistency between the teachings of oneor more of the references incorporated herein and the presentdisclosure, the teachings of the present specification are intended.

What is claimed is:
 1. An IL2 agonist comprising a first polypeptidechain which comprises, in an N- to C-terminal orientation: (a) an IgG Fcdomain comprising a hinge region, wherein the hinge region comprises afirst sequence derived from a first type of IgG and a second sequencederived from a second, different type of IgG; and (b) an IL2 moietycomprising: (i) an IL2-Ra domain comprising an amino acid sequencehaving at least about 90% sequence identity to SEQ ID NO:4; (ii) apeptide linker from 5 to 60 amino acids in length; and (iii) an IL2domain C-terminal to the IL2-Ra domain, wherein the IL2 domain comprisesan amino acid sequence having at least about 90% sequence identity toSEQ ID NO:2.
 2. The IL2 agonist of claim 1, wherein the IL2 domaincomprises an amino acid sequence having at least about 95% sequenceidentity to SEQ ID NO:2.
 3. The IL2 agonist of claim 1, wherein the IL2domain comprises the amino acid sequence of SEQ ID NO:2.
 4. The IL2agonist of claim 1, wherein the IL2-Rα domain comprises the amino acidsequence of SEQ ID NO:4.
 5. The IL2 agonist of claim 1, wherein thepeptide linker is or comprises a glycine-serine linker.
 6. The IL2agonist of claim 1, wherein the Fc domain and the IL2 moiety areconnected via an Fc-IL2 linker.
 7. The IL2 agonist of claim 6, whereinthe Fc-IL2 linker is or comprises a glycine-serine linker.
 8. The IL2agonist of claim 1, wherein the Fc domain is an IgG1 Fc domain.
 9. TheIL2 agonist of claim 1, wherein the Fc domain is an IgG4 Fc domain. 10.The IL2 agonist of claim 1, which is a dimer.
 11. The IL2 agonist ofclaim 10 which comprises a second polypeptide chain, said secondpolypeptide chain comprising in an N- to C-terminal orientation: (a) anIgG Fc domain comprising a hinge region, wherein the hinge regioncomprises a first sequence derived from a first type of IgG and a secondsequence derived from a second, different type of IgG; and (b) an IL2moiety comprising: (i) an IL2-Ra domain comprising an amino acidsequence having at least about 90% sequence identity to SEQ ID NO:4;(ii) a peptide linker from 5 to 60 amino acids in length; and (iii) anIL2 domain C-terminal to the IL2-Ra domain, wherein the IL2 domaincomprises an amino acid sequence having at least about 90% sequenceidentity to SEQ ID NO:2.
 12. The IL2 agonist of claim 11 which is ahomodimer.
 13. A nucleic acid or plurality of nucleic acids encoding theIL2 agonist of claim
 1. 14. A host cell engineered to express the IL2agonist of claim
 1. 15. A pharmaceutical composition comprising the IL2agonist of claim 1 and an excipient.
 16. A method of treating skin,breast, lung or colon cancer, comprising administering to a subject inneed thereof the IL2 agonist of claim
 12. 17. The IL2 agonist of claim1, wherein the peptide linker comprises the amino acid sequence(GGGGS)_(n), wherein n is an integer from 1 to
 10. 18. The IL2 agonistof claim 6, wherein the Fc-IL2 linker comprises the amino acid sequence(GGGGS)_(n), wherein n is an integer from 1 to
 10. 19. The IL2 agonistof claim 1, wherein the IL2-Ra domain comprises an amino acid sequencehaving at least about 95% sequence identity to SEQ ID NO:4.
 20. The IL2agonist of claim 1, wherein the IL2-Ra domain comprises an amino acidsequence having at least about 98% sequence identity to SEQ ID NO:4. 21.The IL2 agonist of claim 1, wherein the IL2 domain comprises an aminoacid sequence having at least about 98% sequence identity to SEQ IDNO:2.
 22. The IL2 agonist of claim 1, wherein the IL2-Ra domaincomprises an amino acid sequence having at least about 95% sequenceidentity to SEQ ID NO:4 and the IL2 domain comprises an amino acidsequence having at least about 95% sequence identity to SEQ ID NO:2. 23.The IL2 agonist of claim 1, wherein the IL2-Ra domain comprises an aminoacid sequence having at least about 98% sequence identity to SEQ ID NO:4and the IL2 domain comprises an amino acid sequence having at leastabout 98% sequence identity to SEQ ID NO:2.
 24. The IL2 agonist of claim1, wherein the IgG Fc domain is an IgG4 Fc domain, the IL2-Ra domaincomprises the amino acid sequence of SEQ ID NO:4, and the IL2 domaincomprises the amino acid sequence of SEQ ID NO:2.
 25. The IL2 agonist ofclaim 24, wherein the Fc domain comprises an amino acid sequence havingat least about 95% sequence identity to residues 99-326 of SEQ ID NO:52.26. The IL2 agonist of claim 24, wherein the Fc domain comprises anamino acid sequence having at least about 98% sequence identity toresidues 99-326 of SEQ ID NO:52.
 27. The IL2 agonist of claim 24,wherein the Fc domain comprises the amino acid sequence of residues99-326 of SEQ ID NO:52.
 28. The IL2 agonist of claim 1, wherein the IL2agonist comprises the amino acid sequence of SEQ ID NO:101.
 29. The IL2agonist of claim 1, wherein the hinge region comprises the amino acidsequence of SEQ ID NO:80.
 30. The IL2 agonist of claim 1, wherein thehinge region comprises the amino acid sequence of SEQ ID NO:81.